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1.
Sci Transl Med ; 12(555)2020 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-32759275

RESUMEN

Obesity is heightened during aging, and although the estrogen receptor α (ERα) has been implicated in the prevention of obesity, its molecular actions in adipocytes remain inadequately understood. Here, we show that adipose tissue ESR1/Esr1 expression inversely associated with adiposity and positively associated with genes involved in mitochondrial metabolism and markers of metabolic health in 700 Finnish men and 100 strains of inbred mice from the UCLA Hybrid Mouse Diversity Panel. To determine the anti-obesity actions of ERα in fat, we selectively deleted Esr1 from white and brown adipocytes in mice. In white adipose tissue, Esr1 controlled oxidative metabolism by restraining the targeted elimination of mitochondria via the E3 ubiquitin ligase parkin. mtDNA content was elevated, and adipose tissue mass was reduced in adipose-selective parkin knockout mice. In brown fat centrally involved in body temperature maintenance, Esr1 was requisite for both mitochondrial remodeling by dynamin-related protein 1 (Drp1) and uncoupled respiration thermogenesis by uncoupled protein 1 (Ucp1). In both white and brown fat of female mice and adipocytes in culture, mitochondrial dysfunction in the context of Esr1 deletion was paralleled by a reduction in the expression of the mtDNA polymerase γ subunit Polg1 We identified Polg1 as an ERα target gene by showing that ERα binds the Polg1 promoter to control its expression in 3T3L1 adipocytes. These findings support strategies leveraging ERα action on mitochondrial function in adipocytes to combat obesity and metabolic dysfunction.

2.
Life Sci Alliance ; 3(7)2020 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-32499316

RESUMEN

Recent breakthroughs in live-cell imaging have enabled visualization of cristae, making it feasible to investigate the structure-function relationship of cristae in real time. However, quantifying live-cell images of cristae in an unbiased way remains challenging. Here, we present a novel, semi-automated approach to quantify cristae, using the machine-learning Trainable Weka Segmentation tool. Compared with standard techniques, our approach not only avoids the bias associated with manual thresholding but more efficiently segments cristae from Airyscan and structured illumination microscopy images. Using a cardiolipin-deficient cell line, as well as FCCP, we show that our approach is sufficiently sensitive to detect perturbations in cristae density, size, and shape. This approach, moreover, reveals that cristae are not uniformly distributed within the mitochondrion, and sites of mitochondrial fission are localized to areas of decreased cristae density. After a fusion event, individual cristae from the two mitochondria, at the site of fusion, merge into one object with distinct architectural values. Overall, our study shows that machine learning represents a compelling new strategy for quantifying cristae in living cells.

3.
Methods Cell Biol ; 155: 545-555, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32183976

RESUMEN

The emergence of diffraction-unlimited live-cell imaging technologies has enabled the examination of mitochondrial form and function in unprecedented detail. We recently developed an approach for visualizing the inner mitochondrial membrane and determined that cristae membranes possess distinct mitochondrial membrane potentials, representing unique bioenergetic subdomains within the same organelle. Here, we outline a methodology for resolving cristae and inner boundary membranes using the LSM880 with Airyscan. Furthermore, we demonstrate how to analyze TMRE fluorescence intensity using the Nernst equation to calculate membrane potentials of individual cristae. Altogether, using these new techniques to study the electrochemical properties of the cristae can help to gain deeper insight into the still elusive nature of the mitochondrion.

4.
EMBO Rep ; 21(3): e49776, 2020 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-32067344

RESUMEN

The mitochondrial inner membrane can reshape under different physiological conditions. How, at which frequency this occurs in living cells, and the molecular players involved are unknown. Here, we show using state-of-the-art live-cell stimulated emission depletion (STED) super-resolution nanoscopy that neighbouring crista junctions (CJs) dynamically appose and separate from each other in a reversible and balanced manner in human cells. Staining of cristae membranes (CM), using various protein markers or two lipophilic inner membrane-specific dyes, further revealed that cristae undergo continuous cycles of membrane remodelling. These events are accompanied by fluctuations of the membrane potential within distinct cristae over time. Both CJ and CM dynamics depended on MIC13 and occurred at similar timescales in the range of seconds. Our data further suggest that MIC60 acts as a docking platform promoting CJ and contact site formation. Overall, by employing advanced imaging techniques including fluorescence recovery after photobleaching (FRAP), single-particle tracking (SPT), live-cell STED and high-resolution Airyscan microscopy, we propose a model of CJ dynamics being mechanistically linked to CM remodelling representing cristae membrane fission and fusion events occurring within individual mitochondria.

5.
EMBO J ; 38(22): e101056, 2019 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-31609012

RESUMEN

The mitochondrial membrane potential (ΔΨm ) is the main driver of oxidative phosphorylation (OXPHOS). The inner mitochondrial membrane (IMM), consisting of cristae and inner boundary membranes (IBM), is considered to carry a uniform ΔΨm . However, sequestration of OXPHOS components in cristae membranes necessitates a re-examination of the equipotential representation of the IMM. We developed an approach to monitor ΔΨm at the resolution of individual cristae. We found that the IMM was divided into segments with distinct ΔΨm , corresponding to cristae and IBM. ΔΨm was higher at cristae compared to IBM. Treatment with oligomycin increased, whereas FCCP decreased, ΔΨm heterogeneity along the IMM. Impairment of cristae structure through deletion of MICOS-complex components or Opa1 diminished this intramitochondrial heterogeneity of ΔΨm . Lastly, we determined that different cristae within the individual mitochondrion can have disparate membrane potentials and that interventions causing acute depolarization may affect some cristae while sparing others. Altogether, our data support a new model in which cristae within the same mitochondrion behave as independent bioenergetic units, preventing the failure of specific cristae from spreading dysfunction to the rest.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Potencial de la Membrana Mitocondrial , Mitocondrias/fisiología , Membranas Mitocondriales/metabolismo , Mioblastos/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/patología , Células Cultivadas , Femenino , Células HeLa , Humanos , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Mitocondriales/metabolismo , Mioblastos/citología , Fosforilación Oxidativa
6.
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.

7.
J Mol Biol ; 430(24): 4823-4833, 2018 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-30389435

RESUMEN

Elamipretide is a tetrapeptide that restores defects in mitochondrial function, binds to cardiolipin, and is being tested in clinical trials for mitochondria-related diseases. However, whether elamipretide modulates mitochondrial quality control and dynamics, processes essential to preserve mitochondrial function, is unclear. Thus, we tested the effects of elamipretide on mitochondrial morphology, mitophagosome formation, and their early disruption induced by excess nutrients in INS1 ß-cells. Elamipretide treatment was sufficient to increase engulfment of mitochondria into autophagosomes in control INS1 ß-cells, without inducing widespread changes in mitochondrial morphology or membrane potential. In an early pathogenic context mimicked by short-term exposure to nutrient excess, elamipretide treatment prevented both mitochondrial fragmentation and defects in the engulfment of mitochondria into autophagosomes. On the other hand, elamipretide did not prevent lysosomal defects induced by nutrient excess. Accordingly, elamipretide treatment did not entail benefits on pathogenic p62 and LC3II accumulation or on insulin secretory function. In conclusion, our data show that elamipretide selectively stimulates the engulfment of mitochondria into autophagosomes and prevents its defects induced by nutrient excess. Thus, we propose that improved selectivity of mitochondrial quality control processes might contribute to the benefits stemming from elamipretide treatments in other disease models.


Asunto(s)
Autofagosomas/metabolismo , Células Secretoras de Insulina/citología , Mitocondrias/efectos de los fármacos , Nutrientes/farmacología , Oligopéptidos/farmacología , Línea Celular , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Lisosomas/metabolismo , Potencial de la Membrana Mitocondrial , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/metabolismo , Recambio Mitocondrial/efectos de los fármacos , Proteínas de Unión al ARN/metabolismo
8.
iScience ; 5: 99-109, 2018 Jul 27.
Artículo en Inglés | MEDLINE | ID: mdl-30240649

RESUMEN

B lymphocytes provide adaptive immunity by generating antigen-specific antibodies and supporting the activation of T cells. Little is known about how global metabolism supports naive B cell activation to enable an effective immune response. By coupling RNA sequencing (RNA-seq) data with glucose isotopomer tracing, we show that stimulated B cells increase programs for oxidative phosphorylation (OXPHOS), the tricarboxylic acid (TCA) cycle, and nucleotide biosynthesis, but not glycolysis. Isotopomer tracing uncovered increases in TCA cycle intermediates with almost no contribution from glucose. Instead, glucose mainly supported the biosynthesis of ribonucleotides. Glucose restriction did not affect B cell functions, yet the inhibition of OXPHOS or glutamine restriction markedly impaired B cell growth and differentiation. Increased OXPHOS prompted studies of mitochondrial dynamics, which revealed extensive mitochondria remodeling during activation. Our results show how B cell metabolism adapts with stimulation and reveals unexpected details for carbon utilization and mitochondrial dynamics at the start of a humoral immune response.

9.
Mol Metab ; 16: 150-159, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30098928

RESUMEN

OBJECTIVE: Islets from the same pancreas show remarkable variability in glucose sensitivity. While mitochondrial respiration is essential for glucose-stimulated insulin secretion, little is known regarding heterogeneity in mitochondrial function at the individual islet level. This is due in part to a lack of high-throughput and non-invasive methods for detecting single islet function. METHODS: We have developed a novel non-invasive, high-throughput methodology capable of assessing mitochondrial respiration in large-sized individual islets using the XF96 analyzer (Agilent Technologies). RESULTS: By increasing measurement sensitivity, we have reduced the minimal size of mouse and human islets needed to assess mitochondrial respiration to single large islets of >35,000 µm2 area (∼210 µm diameter). In addition, we have measured heterogeneous glucose-stimulated mitochondrial respiration among individual human and mouse islets from the same pancreas, allowing population analyses of islet mitochondrial function for the first time. CONCLUSIONS: We have developed a novel methodology capable of analyzing mitochondrial function in large-sized individual islets. By highlighting islet functional heterogeneity, we hope this methodology can significantly advance islet research.


Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Islotes Pancreáticos/metabolismo , Páncreas/metabolismo , Adulto , Anciano , Animales , Respiración de la Célula/fisiología , Femenino , Glucosa/metabolismo , Ensayos Analíticos de Alto Rendimiento/instrumentación , Humanos , Insulina/metabolismo , Secreción de Insulina/fisiología , Islotes Pancreáticos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Mitocondrias/metabolismo , Cultivo Primario de Células
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