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1.
iScience ; 23(5): 101125, 2020 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-32428862

RESUMEN

Increased flux of glucose through glycolysis is a hallmark of inflammatory macrophages and is essential for optimal effector functions. Solute carrier (SLC) 37A2 is an endoplasmic reticulum-anchored phosphate-linked glucose-6-phosphate transporter that is highly expressed in macrophages and neutrophils. We demonstrate that SLC37A2 plays a pivotal role in murine macrophage inflammatory activation and cellular metabolic rewiring. Toll-like receptor (TLR) 4 stimulation by lipopolysaccharide (LPS) rapidly increases macrophage SLC37A2 protein expression. SLC37A2 deletion reprograms macrophages to a hyper-glycolytic process and accelerates LPS-induced inflammatory cytokine production, which partially depends on nicotinamide adenine dinucleotide (NAD+) biosynthesis. Blockade of glycolysis normalizes the differential expression of pro-inflammatory cytokines between control and SLC37A2 deficient macrophages. Conversely, overexpression of SLC37A2 lowers macrophage glycolysis and significantly reduces LPS-induced pro-inflammatory cytokine expression. In conclusion, our study suggests that SLC37A2 dampens murine macrophage inflammation by down-regulating glycolytic reprogramming as a part of macrophage negative feedback system to curtail acute innate activation.

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.
J Nutr ; 149(9): 1493-1502, 2019 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-31112997

RESUMEN

BACKGROUND: Western diets are associated with increased incidences of obesity, hypertension, diabetes, and hypercholesterolemia, whereas Mediterranean diets, richer in polyphenols, monounsaturated fats, fruits, vegetables, poultry, and fish, appear to have cardiometabolic health benefits. Previous work has included population-based studies with limited evidence for causation or animal studies focused on single macro- or micronutrients; therefore, primate animal models provide an opportunity to determine potential mechanisms underlying the effects of dietary patterns on health and disease. OBJECTIVE: The aim of this study was to determine the effects of whole dietary patterns, either a Western or Mediterranean diet, on skeletal muscle mitochondrial bioenergetics in cynomolgus macaques. METHODS: In this study, 22 adult female cynomolgus macaques (∼11-14 y by dentition) were fed either a Western or Mediterranean diet for 30 mo. The Western diet was designed to mimic the diet of a middle-aged American woman and the Mediterranean diet included key aspects of Mediterranean diets studied in humans, such as plant-based proteins and fat, complex carbohydrates, and fiber. Diets were matched on macronutrient composition (16% protein, 54% carbohydrate, and 31% fat) and cholesterol content. Skeletal muscle was collected for high-resolution respirometry, citrate synthase activity, and western blot measurements. Pearson correlation analysis between respirometry measures and measures of carbohydrate metabolism was also performed. RESULTS: We found that consumption of a Western diet resulted in significantly higher mitochondrial respiration with fatty acid oxidation (FAO) (53%), FAO + complex I (52%), complex I + II (31%), max electron transport system (ETS) (31%), and ETS rotenone sensitive (31%) than did consumption of a Mediterranean diet. In addition, measures of respiration in response to fatty acids were significantly and positively correlated with both insulin resistance and plasma insulin concentrations. CONCLUSIONS: This study highlights the importance of dietary composition in mitochondrial bioenergetics and that diet can influence skeletal muscle mitochondrial respiration independently of other factors such as macronutrient composition.

4.
Clin Sci (Lond) ; 132(23): 2509-2518, 2018 12 12.
Artículo en Inglés | MEDLINE | ID: mdl-30401689

RESUMEN

Blood-based bioenergetic profiling has promising applications as a minimally invasive biomarker of systemic bioenergetic capacity. In the present study, we examined peripheral blood mononuclear cell (PBMC) mitochondrial function and brain morphology in a cohort of African Americans with long-standing Type 2 diabetes. Key parameters of PBMC respiration were correlated with white matter, gray matter, and total intracranial volumes. Our analyses indicate that these relationships are primarily driven by the relationship of systemic bioenergetic capacity with total intracranial volume, suggesting that systemic differences in mitochondrial function may play a role in overall brain morphology.


Asunto(s)
Afroamericanos , Encéfalo/diagnóstico por imagen , Complicaciones de la Diabetes/sangre , Diabetes Mellitus Tipo 2/sangre , Metabolismo Energético , Leucocitos Mononucleares/metabolismo , Imagen por Resonancia Magnética , Mitocondrias/metabolismo , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Estudios Transversales , Complicaciones de la Diabetes/diagnóstico por imagen , Complicaciones de la Diabetes/etnología , Diabetes Mellitus Tipo 2/diagnóstico por imagen , Diabetes Mellitus Tipo 2/etnología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas
5.
Sci Rep ; 8(1): 6635, 2018 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-29703899

RESUMEN

Mitochondrial reactive oxygen species (ROS) are essential regulators of cellular signaling, metabolism and epigenetics underlying the pathophysiology of numerous diseases. Despite the critical function of redox regulation in mitochondria, currently there are limited methods available to monitor protein oxidation in this key subcellular organelle. Here, we describe compounds for imaging sulfenylated proteins in mitochondria: DCP-NEt2-Coumarin (DCP-NEt2C) and rhodamine-based DCP-Rho1. Side-by-side comparison studies are presented on the reactivity of DCP-NEt2C and DCP-Rho1 with a model protein sulfenic acid (AhpC-SOH) and mitochondrial localization to identify optimized experimental conditions for labeling and visualization of protein sulfenylation that would be independent of mitochondria membrane potential and would not impact mitochondrial function. These probes are applied to image mitochondrial protein sulfenylation under conditions of serum starvation and in a cell culture model of lung cancer exposed to ionizing radiation and silver nanoparticles, agents serving dual functions as environmental stressors and cancer therapeutics.


Asunto(s)
Técnicas Citológicas/métodos , Indicadores y Reactivos/síntesis química , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Sondas Moleculares/síntesis química , Procesamiento Proteico-Postraduccional , Coloración y Etiquetado/métodos , Células A549 , Humanos , Oxidación-Reducción , Ácidos Sulfénicos/metabolismo
6.
Oxid Med Cell Longev ; 2017: 7317251, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29098063

RESUMEN

Blood-based bioenergetic profiling provides a minimally invasive assessment of mitochondrial health shown to be related to key features of aging. Previous studies show that blood cells recapitulate mitochondrial alterations in the central nervous system under pathological conditions, including the development of Alzheimer's disease. In this study of nonhuman primates, we focus on mitochondrial function and bioenergetic capacity assessed by the respirometric profiling of monocytes, platelets, and frontal cortex mitochondria. Our data indicate that differences in the maximal respiratory capacity of brain mitochondria are reflected by CD14+ monocyte maximal respiratory capacity and platelet and monocyte bioenergetic health index. A subset of nonhuman primates also underwent [18F] fluorodeoxyglucose positron emission tomography (FDG-PET) imaging to assess brain glucose metabolism. Our results indicate that platelet respiratory capacity positively correlates to measures of glucose metabolism in multiple brain regions. Altogether, the results of this study provide early evidence that blood-based bioenergetic profiling is related to brain mitochondrial metabolism. While these measures cannot substitute for direct measures of brain metabolism, provided by measures such as FDG-PET, they may have utility as a metabolic biomarker and screening tool to identify individuals exhibiting systemic bioenergetic decline who may therefore be at risk for the development of neurodegenerative diseases.


Asunto(s)
Encéfalo/fisiopatología , Metabolismo Energético/genética , Animales , Femenino , Haplorrinos , Humanos
8.
Obesity (Silver Spring) ; 25(4): 689-696, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-28236433

RESUMEN

OBJECTIVE: Obesity exists with and without accompanying cardiometabolic disease, termed metabolically unhealthy obesity (MUO) and healthy obesity (MHO), respectively. Underlying differences in the ability of subcutaneous (SQ) fat to respond to nutrient excess are emerging as a key pathway. This study aimed to document the first spontaneous animal model of MHO and MUO and differences in SQ adipose tissue. METHODS: Vervet monkeys (Chlorocebus aethiops; N = 171) were screened for metabolic syndrome. A subset of MHO and MUO monkeys (n = 6/group) had SQ fat biopsies collected for histological evaluations and examination of key mitochondrial proteins. RESULTS: Obesity was seen in 20% of monkeys, and within this population, 31% were healthy, which mirrors human prevalence estimates. MUO monkeys had more than 60% lower adiponectin concentrations despite similar fat cell size, uncoupling protein 3, and activated macrophage abundance. However, alternatively activated/anti-inflammatory macrophages were 70% lower. Deficiencies of 50% or more in mitochondrial quality control regulators and selected mitochondrial fission and fusion markers were observed in the SQ fat of MUO monkeys despite comparable mitochondrial content. CONCLUSIONS: A novel and translatable spontaneously obese animal model of MHO and MUO, occurring independently of dietary factors, was characterized. Differences in mitochondrial quality and inflammatory cell populations of subcutaneous fat may underpin divergent metabolic health.


Asunto(s)
Síndrome Metabólico/fisiopatología , Mitocondrias/metabolismo , Obesidad/fisiopatología , Grasa Subcutánea/metabolismo , Adiponectina/análisis , Animales , Biomarcadores/análisis , Chlorocebus aethiops , Modelos Animales de Enfermedad , Activación de Macrófagos , Macrófagos/metabolismo , Grasa Subcutánea/citología , Proteína Desacopladora 3/análisis
9.
J Am Soc Nephrol ; 28(4): 1093-1105, 2017 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-27821631

RESUMEN

APOL1 G1 and G2 variants facilitate kidney disease in blacks. To elucidate the pathways whereby these variants contribute to disease pathogenesis, we established HEK293 cell lines stably expressing doxycycline-inducible (Tet-on) reference APOL1 G0 or the G1 and G2 renal-risk variants, and used Illumina human HT-12 v4 arrays and Affymetrix HTA 2.0 arrays to generate global gene expression data with doxycycline induction. Significantly altered pathways identified through bioinformatics analyses involved mitochondrial function; results from immunoblotting, immunofluorescence, and functional assays validated these findings. Overexpression of APOL1 by doxycycline induction in HEK293 Tet-on G1 and G2 cells led to impaired mitochondrial function, with markedly reduced maximum respiration rate, reserve respiration capacity, and mitochondrial membrane potential. Impaired mitochondrial function occurred before intracellular potassium depletion or reduced cell viability occurred. Analysis of global gene expression profiles in nondiseased primary proximal tubule cells from black patients revealed that the nicotinate phosphoribosyltransferase gene, responsible for NAD biosynthesis, was among the top downregulated transcripts in cells with two APOL1 renal-risk variants compared with those without renal-risk variants; nicotinate phosphoribosyltransferase also displayed gene expression patterns linked to mitochondrial dysfunction in HEK293 Tet-on APOL1 cell pathway analyses. These results suggest a pivotal role for mitochondrial dysfunction in APOL1-associated kidney disease.


Asunto(s)
Apolipoproteínas/genética , Enfermedades Renales/genética , Lipoproteínas HDL/genética , Enfermedades Mitocondriales/genética , Grupo de Ascendencia Continental Africana , Apolipoproteína L1 , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Células HEK293 , Humanos , Masculino , Persona de Mediana Edad , Factores de Riesgo
10.
Redox Biol ; 10: 65-77, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27693859

RESUMEN

Blood based bioenergetic profiling strategies are emerging as potential reporters of systemic mitochondrial function; however, the extent to which these measures reflect the bioenergetic capacity of other tissues is not known. The premise of this work is that highly metabolically active tissues, such as skeletal and cardiac muscle, are susceptible to differences in systemic bioenergetic capacity. Therefore, we tested whether the respiratory capacity of blood cells, monocytes and platelets, are related to contemporaneous respirometric assessments of skeletal and cardiac muscle mitochondria. 18 female vervet/African green monkeys (Chlorocebus aethiops sabaeus) of varying age and metabolic status were examined for this study. Monocyte and platelet maximal capacity correlated with maximal oxidative phosphorylation capacity of permeabilized skeletal muscle (R=0.75, 95% confidence interval [CI]: 0.38-0.97; R=0.51, 95%CI: 0.05-0.81; respectively), isolated skeletal muscle mitochondrial respiratory control ratio (RCR; R=0.70, 95%CI: 0.35-0.89; R=0.64, 95%CI: 0.23-0.98; respectively), and isolated cardiac muscle mitochondrial RCR (R=0.55, 95%CI: 0.22-0.86; R=0.58, 95%CI: 0.22-0.85; respectively). These results suggest that blood based bioenergetic profiling may be used to report on the bioenergetic capacity of muscle tissues. Blood cell respirometry represents an attractive alternative to tissue based assessments of mitochondrial function in human studies based on ease of access and the minimal participant burden required by these measures.


Asunto(s)
Plaquetas/metabolismo , Metabolismo Energético , Mitocondrias Cardíacas/metabolismo , Mitocondrias Musculares/metabolismo , Monocitos/metabolismo , Animales , Biomarcadores/metabolismo , Respiración de la Célula , Chlorocebus aethiops , Femenino , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Fosforilación Oxidativa , Consumo de Oxígeno
11.
JACC Heart Fail ; 4(8): 636-45, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27179829

RESUMEN

OBJECTIVES: The aim of this study was to examine skeletal muscle mitochondria content, oxidative capacity, and the expression of key mitochondrial dynamics proteins in patients with heart failure with preserved ejection fraction (HFpEF), as well as to determine potential relationships with measures of exercise performance. BACKGROUND: Multiple lines of evidence indicate that severely reduced peak exercise oxygen uptake (peak VO2) in older patients with HFpEF is related to abnormal skeletal muscle oxygen utilization. Mitochondria are key regulators of skeletal muscle metabolism; however, little is known about how these organelles are affected in HFpEF. METHODS: Both vastus lateralis skeletal muscle citrate synthase activity and the expression of porin and regulators of mitochondrial fusion were examined in older patients with HFpEF (n = 20) and healthy, age-matched control subjects (n = 17). RESULTS: Compared with age-matched healthy control subjects, mitochondrial content assessed by porin expression was 46% lower (p = 0.01), citrate synthase activity was 29% lower (p = 0.01), and Mfn2 (mitofusin 2) expression was 54% lower (p <0.001) in patients with HFpEF. Expression of porin was significantly positively correlated with both peak VO2 and 6-min walk distance (r = 0.48, p = 0.003 and r = 0.33, p = 0.05, respectively). Expression of Mfn2 was also significantly positively correlated with both peak VO2 and 6-min walk distance (r = 0.40, p = 0.02 and r = 0.37, p = 0.03 respectively). CONCLUSIONS: These findings suggest that skeletal muscle oxidative capacity, mitochondrial content, and mitochondrial fusion are abnormal in older patients with HFpEF and might contribute to their severe exercise intolerance.


Asunto(s)
Tolerancia al Ejercicio/fisiología , GTP Fosfohidrolasas/metabolismo , Insuficiencia Cardíaca/metabolismo , Mitocondrias Musculares/metabolismo , Proteínas Mitocondriales/metabolismo , Consumo de Oxígeno , Músculo Cuádriceps/metabolismo , Volumen Sistólico , Factores de Edad , Anciano , Estudios de Casos y Controles , Citrato (si)-Sintasa/metabolismo , Femenino , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Dinámicas Mitocondriales , Músculo Cuádriceps/patología , Canal Aniónico 1 Dependiente del Voltaje/metabolismo
12.
BMC Obes ; 2: 40, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26448868

RESUMEN

BACKGROUND: Mitochondrial function declines with age; however, the relationship between adiposity and mitochondrial function among older adults is unclear. This study examined relationships between skeletal muscle mitochondrial content and electron transport chain complex 2 driven respiration with whole body and thigh composition, body fat distribution, and insulin sensitivity in older adults. METHODS: 25 healthy, sedentary, weight-stable men (N = 13) and women (N = 12) >65 years of age, with a BMI range of 18-35 kg/m(2), participated in this study. Vastus lateralis biopsies were analyzed for citrate synthase (CS) activity and succinate mediated respiration of isolated mitochondria. Whole body and thigh composition were measured by DXA and CT. HOMA-IR was calculated using fasting glucose and insulin as an estimate of insulin sensitivity. RESULTS: Similar to reports in middle-aged adults, skeletal muscle CS activity was negatively correlated with BMI (R = -0.43) in our cohort of older adults. Higher total and thigh adiposity were correlated with lower CS activity independent of BMI (R = -0.50 and -0.71 respectively). Maximal complex 2 driven mitochondrial respiration was negatively correlated with lower body adiposity in males (R = -0.66). In this cohort of non-diabetic older adults, both HOMA-IR and insulin were positively correlated with CS activity when controlling for BMI (R = 0.57 and 0.66 respectively). CONCLUSIONS: Adiposity and body composition are correlated with skeletal muscle mitochondrial content and electron transport chain function in healthy, sedentary, community dwelling, older adults. Specific relationships of mitochondrial bioenergetics with gender and insulin sensitivity are also apparent. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT01049698.

13.
Exp Gerontol ; 70: 84-91, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26226578

RESUMEN

BACKGROUND: Physical function and strength decline with age and lead to limited mobility and independence in older adults. Alterations in mitochondrial function are thought to underlie numerous age-related changes, including declining physical ability. Recent studies suggest that systemic changes in bioenergetic capacity may be reported by analyzing mitochondrial function in circulating cells. The objective of this study was to determine whether the bioenergetic capacity of peripheral blood mononuclear cells (PBMCs) is related to differences in physical function among older, overweight/obese, adults. To address this, we tested the hypothesis that greater PBMC respirometric capacity would be associated with better physical function, muscular strength, leg lean mass, and muscle quality. Furthermore, we tested whether the respirometric capacity of PBMCs is related to cellular composition and inflammatory status reported by interleukin-6 (IL-6). METHODS: Fasted PBMC respiration (pmol/min/500,000 cells), expanded short physical performance battery (Ex-SPPB), peak knee extensor (KE) strength (Nm), grip strength (kg), leg lean mass (kg, via dual energy X-ray absorptiometry [DXA]), muscle quality (Nm/kg), and plasma IL-6 (pg/mL) were analyzed in 15 well-functioning, community-dwelling, sedentary overweight/obese older men (n=9) and women (n=6) aged 65 to 78 (mean 68.3 ± 3.5 years). Pearson and partial correlations were calculated to determine associations between PBMC respiration and these variables. RESULTS: Higher maximal respiration of PBMCs was associated with better Ex-SPPB (r=0.58, p=0.02), greater KE strength (r=0.60, p=0.02), greater grip strength (r=0.52, p=0.05) and lower IL-6 (r=-0.58, p=0.04). Higher spare respiratory capacity was associated with better Ex-SPPB (r=0.59, p=0.02), greater KE strength (r=0.60, p=0.02), greater grip strength (r=0.54, p=0.04), greater leg muscle quality (r=0.56, p=0.04), and lower IL-6 (r=-0.55, p=0.05). Monocyte and lymphocyte counts were not related to PBMC respiratory capacity. CONCLUSIONS: Our results indicate that respirometric profiles of readily obtainable blood cells are associated with physical function and strength. Future studies should be undertaken in order to determine whether blood-based bioenergetic profiling can provide an objective index of systemic mitochondrial health.


Asunto(s)
Inflamación/sangre , Leucocitos Mononucleares/fisiología , Sobrepeso/sangre , Aptitud Física/fisiología , Anciano , Biomarcadores/sangre , Respiración de la Célula/fisiología , Metabolismo Energético/fisiología , Prueba de Esfuerzo/métodos , Femenino , Humanos , Inflamación/fisiopatología , Interleucina-6/sangre , Recuento de Leucocitos , Masculino , Fuerza Muscular/fisiología , Músculo Esquelético/fisiopatología , Obesidad/sangre , Obesidad/fisiopatología , Sobrepeso/fisiopatología
14.
J Vis Exp ; (96)2015 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-25741892

RESUMEN

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 µg of mitochondria indicate that 1.0 µg is sufficient to measure respiration and that 5.0 µg provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.


Asunto(s)
Biopsia con Aguja/métodos , Mitocondrias Musculares/química , Mitocondrias Musculares/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Consumo de Oxígeno/fisiología , Humanos , Oxidación-Reducción
15.
Radiat Res ; 183(3): 291-304, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25738895

RESUMEN

While radiation therapy is commonly used for treating cancer, radiation resistance can limit long-term control of the disease. In this study, we investigated the reprogramming of the energy metabolism in radiosensitive and radioresistant head and neck squamous cell carcinomas (HNSCC) using a preclinical matched model of radiation resistance. Our investigation found that radioresistant rSCC-61 cells: 1. They display increased glucose uptake and decreased fatty acid uptake; 2. They deviate from the classical Warburg effect by diverting the glycolytic flux into the pentose phosphate pathway; 3. They are more dependent on glucose than glutamine metabolism to support growth; 4. They have decreased mitochondrial oxidative phosphorylation; 5. They have enhanced fatty acid biosynthesis by increasing the expression of fatty acid synthase; and 6. They utilize endogenous fatty acids to meet the energy demands for proliferation. Inhibition of fatty acid synthase with orlistat or FASN siRNA resulted in increased cytotoxicity and sensitivity to radiation in rSCC-61 cells. These results demonstrate the potential of combination therapy using radiation and orlistat or other inhibitors of lipid and energy metabolism for treating radiation resistance in HNSCC.


Asunto(s)
Metabolismo Energético , Neoplasias de Cabeza y Cuello/radioterapia , Neoplasias de Células Escamosas/radioterapia , Tolerancia a Radiación , Ácido Graso Sintasas/antagonistas & inhibidores , Glucosa/metabolismo , Glucólisis , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Neoplasias de Células Escamosas/metabolismo
16.
J Gerontol A Biol Sci Med Sci ; 70(11): 1394-9, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25030980

RESUMEN

BACKGROUND: Gait speed provides an integrated measure of physical ability that is predictive of morbidity, disability, and mortality in older adults. Energy demands associated with walking suggest that mitochondrial bioenergetics may play a role in gait speed. Here, we examined the relationship between gait speed and skeletal muscle mitochondrial bioenergetics, and further evaluated whether blood-based bioenergetic profiling might have similar associations with gait speed. METHODS: Participants in this study were comprised of two subsets (n = 17 per subset) and were overweight/obese (body mass index, 30.9 ± 2.37), well-functioning, community-dwelling older adults (69.1 ± 3.69 years) without major comorbidity. Gait speeds were calculated from a fast-paced 400 m walk test. Respiratory control ratios were measured from mitochondria isolated from leg skeletal muscle biopsies from one subset. Maximal respiration and spare respiratory capacity were measured from peripheral blood mononuclear cells from the other subset. RESULTS: Individual differences in gait speed correlated directly with respiratory control ratio of mitochondria isolated from skeletal muscle (r = .536, p = .027) and with both maximal respiration and spare respiratory capacity of peripheral blood mononuclear cells (r = .585 and p = .014; r = .609 and p = .009, respectively). CONCLUSIONS: The bioenergetic profile of mitochondria isolated from skeletal muscle is associated with gait speed in older adults. Blood-based bioenergetic profiling is also associated with gait speed and may provide an alternative measure of mitochondrial function.


Asunto(s)
Metabolismo Energético/fisiología , Marcha/fisiología , Leucocitos Mononucleares/fisiología , Mitocondrias Musculares/fisiología , Músculo Esquelético/fisiología , Consumo de Oxígeno/fisiología , Factores de Edad , Anciano , Respiración de la Célula/fisiología , Femenino , Evaluación Geriátrica , Humanos , Masculino
17.
J Biol Chem ; 290(1): 396-408, 2015 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-25404738

RESUMEN

We reported that NAD(+)-dependent SIRT1, RELB, and SIRT6 nuclear proteins in monocytes regulate a switch from the glycolysis-dependent acute inflammatory response to fatty acid oxidation-dependent sepsis adaptation. We also found that disrupting SIRT1 activity during adaptation restores immunometabolic homeostasis and rescues septic mice from death. Here, we show that nuclear SIRT1 guides RELB to differentially induce SIRT3 expression and also increases mitochondrial biogenesis, which alters bioenergetics during sepsis adaptation. We constructed this concept using TLR4-stimulated THP1 human promonocytes, a model that mimics the initiation and adaptation stages of sepsis. Following increased expression, mitochondrial SIRT3 deacetylase activates the rate-limiting tricarboxylic acid cycle (TCA) isocitrate dehydrogenase 2 and superoxide dismutase 2, concomitant with increases in citrate synthase activity. Mitochondrial oxygen consumption rate increases early and decreases during adaptation, parallel with modifications to membrane depolarization, ATP generation, and production of mitochondrial superoxide and whole cell hydrogen peroxide. Evidence of SIRT1-RELB induction of mitochondrial biogenesis included increases in mitochondrial mass, mitochondrial-to-nuclear DNA ratios, and both nuclear and mitochondrial encoded proteins. We confirmed the SIRT-RELB-SIRT3 adaptation link to mitochondrial bioenergetics in both TLR4-stimulated normal and sepsis-adapted human blood monocytes and mouse splenocytes. We also found that SIRT1 inhibition ex vivo reversed the sepsis-induced changes in bioenergetics.


Asunto(s)
Núcleo Celular/metabolismo , Mitocondrias/metabolismo , Monocitos/metabolismo , Sepsis/metabolismo , Sirtuina 1/metabolismo , Sirtuina 3/metabolismo , Factor de Transcripción ReIB/metabolismo , Animales , Línea Celular , Núcleo Celular/inmunología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Glucólisis/efectos de los fármacos , Humanos , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/inmunología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/patología , Fosforilación Oxidativa/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Cultivo Primario de Células , Sepsis/genética , Sepsis/inmunología , Sepsis/patología , Transducción de Señal , Sirtuina 1/genética , Sirtuina 3/genética , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/metabolismo , Bazo/patología , Factor de Transcripción ReIB/genética
18.
Redox Biol ; 2: 1008-15, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25460737

RESUMEN

Inner mitochondrial membrane peptidase 2-like (IMMP2L) protein is a mitochondrial inner membrane peptidase that cleaves the signal peptide sequences of cytochrome c1 (CYC1) and mitochondrial glycerol phosphate dehydrogenase (GPD2). Immp2l mutant mice show infertility and early signs of aging. It is unclear whether mitochondrial respiratory deficiency underlies this phenotype. Here we show that the intermediate forms of GPD2 and CYC1 have normal expression levels and enzymatic function in Immp2l mutants. Mitochondrial respiration is not diminished in isolated mitochondria and cells from mutant mice. Our data suggest that respiratory deficiency is not the cause of the observed Immp2l mutant phenotypes.


Asunto(s)
Endopeptidasas/metabolismo , Metabolismo Energético , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Mutación , Consumo de Oxígeno , Animales , Endopeptidasas/genética , Ratones , Ratones Mutantes , Mitocondrias/genética , Proteínas Mitocondriales/genética
19.
J Vis Exp ; (65): e3991, 2012 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-22847388

RESUMEN

Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment. Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer's disease, Parkinson's disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function. Mitochondrial fission has been shown to signal a cell to progress toward apoptosis. It can also provide a mechanism by which to separate depolarized and inactive mitochondria to keep the bulk of the network robust. Fusion of mitochondria, on the other hand, leads to sharing of matrix proteins, solutes, mtDNA and the electrochemical gradient, and also seems to prevent progression to apoptosis. How fission and fusion of mitochondria affects cell homeostasis and ultimately the functioning of the organism needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary. Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. The hybrid PEG fusion assay, mixes two populations of differently labeled cells (mtRFP and mtYFP), and analyzes the amount of mixing and colocalization of fluorophores in fused, multinucleated, cells. Although this method has yielded valuable information, not all cell types can fuse, and the conditions under which fusion is stimulated involves the use of toxic drugs that likely affect the normal fusion process. More recently, a cell free technique has been devised, using isolated mitochondria to observe fusion events based on a luciferase assay. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment. A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP). Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the TMRE dye. Mitochondria that depolarize no longer have a sufficient membrane potential and tend not to fuse as efficiently if at all. Therefore, active fusing mitochondria can be tracked with these low levels of TMRE. Accumulation of depolarized mitochondria that lack a TMRE signal may be a sign of phototoxicity or cell death. Higher concentrations of TMRE render mitochondria very sensitive to laser light, and therefore great care must be taken to avoid overlabeling with TMRE. If the effect of depolarization of mitochondria is the topic of interest, a technique using slightly higher levels of TMRE and more intense laser light can be used to depolarize mitochondria in a controlled fashion (Mitra and Lippincott-Schwartz, 2010). To ensure that toxicity due to TMRE is not an issue, we suggest exposing loaded cells (3-15 nM TMRE) to the imaging parameters that will be used in the assay (perhaps 7 stacks of 6 optical sections in a row), and assessing cell health after 2 hours. If the mitochondria appear too fragmented and cells are dying, other mitochondrial markers, such as dsRED or Mitotracker red could be used instead of TMRE. The mtPAGFP method has revealed details about mitochondrial network behavior that could not be visualized using other methods. For example, we now know that mitochondrial fusion can be full or transient, where matrix content can mix without changing the overall network morphology. Additionally, we know that the probability of fusion is independent of contact duration and organelle dimension, is influenced by organelle motility, membrane potential and history of previous fusion activity. In this manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15 nM TMRE in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.


Asunto(s)
Proteínas Fluorescentes Verdes/química , Microscopía Confocal/métodos , Mitocondrias/química , Fusión Celular , Línea Celular , Humanos , Mitocondrias/fisiología , Compuestos Organometálicos/química
20.
Invest Ophthalmol Vis Sci ; 52(12): 8657-64, 2011 Nov 07.
Artículo en Inglés | MEDLINE | ID: mdl-21979999

RESUMEN

PURPOSE: Mitochondrial dysfunction is known to play a role in retinal vascular cell loss, a prominent lesion of diabetic retinopathy. High glucose (HG) has been reported to induce mitochondrial fragmentation and dysfunction in retinal endothelial cells, contributing to apoptosis. In this study, the effects of HG on mitochondrial morphology, membrane potential, and metabolic changes and whether they could contribute to HG-induced apoptosis in retinal pericytes were investigated. METHODS: Bovine retinal pericytes (BRPs) were grown in normal or HG medium for 7 days. Both sets of cells were double stained with mitochondrial membrane potential-independent dye and tetramethylrhodamine-ethyl-ester-perchlorate (TMRE) and imaged by confocal microscopy. The images were analyzed for average mitochondria shape, by using form factor and aspect ratio values, and membrane potential changes, by using the ratio between the red and green dye. BRPs grown in normal or HG medium were analyzed for transient changes in oxygen consumption and extracellular acidification with a flux analyzer and apoptosis by TUNEL assay. RESULTS: BRPs grown in HG media exhibited significant fragmentation of mitochondria and increased membrane potential heterogeneity compared with the BRPs grown in normal medium. Concomitantly, BRPs grown in HG showed reduced steady state and maximum oxygen consumption and reduced extracellular acidification. Number of TUNEL-positive pericytes was increased in HG condition as well. CONCLUSIONS: In HG condition, mitochondria of retinal pericytes display significant fragmentation, metabolic dysfunction, and reduced extracellular acidification. The detrimental effects of HG on mitochondrial function and cellular metabolism could play a role in the accelerated apoptosis associated with the retinal pericytes in diabetic retinopathy.


Asunto(s)
Retinopatía Diabética/fisiopatología , Glucosa/farmacología , Hiperglucemia/fisiopatología , Mitocondrias/fisiología , Pericitos/metabolismo , Vasos Retinianos/citología , Ácidos/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Bovinos , Células Cultivadas , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/fisiología , Hiperglucemia/metabolismo , Hiperglucemia/patología , Etiquetado Corte-Fin in Situ , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Consumo de Oxígeno/efectos de los fármacos , Consumo de Oxígeno/fisiología , Pericitos/efectos de los fármacos , Pericitos/patología
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