RESUMEN
PGC1α is a key transcriptional coregulator of oxidative metabolism and thermogenesis. Through a high-throughput chemical screen, we found that molecules antagonizing the TRPVs (transient receptor potential vanilloid), a family of ion channels, induced PGC1α expression in adipocytes. In particular, TRPV4 negatively regulated the expression of PGC1α, UCP1, and cellular respiration. Additionally, it potently controlled the expression of multiple proinflammatory genes involved in the development of insulin resistance. Mice with a null mutation for TRPV4 or wild-type mice treated with a TRPV4 antagonist showed elevated thermogenesis in adipose tissues and were protected from diet-induced obesity, adipose inflammation, and insulin resistance. This role of TRPV4 as a cell-autonomous mediator for both the thermogenic and proinflammatory programs in adipocytes could offer a target for treating obesity and related metabolic diseases.
Asunto(s)
Metabolismo Energético , Canales Catiónicos TRPV/metabolismo , Termogénesis , Adipocitos/metabolismo , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Femenino , Técnicas de Silenciamiento del Gen , Canales Iónicos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Mitocondriales/metabolismo , Obesidad/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/genética , Transactivadores/metabolismo , Factores de Transcripción , Proteína Desacopladora 1RESUMEN
Brown fat generates heat via the mitochondrial uncoupling protein UCP1, defending against hypothermia and obesity. Recent data suggest that there are two distinct types of brown fat: classical brown fat derived from a myf-5 cellular lineage and UCP1-positive cells that emerge in white fat from a non-myf-5 lineage. Here, we report the isolation of "beige" cells from murine white fat depots. Beige cells resemble white fat cells in having extremely low basal expression of UCP1, but, like classical brown fat, they respond to cyclic AMP stimulation with high UCP1 expression and respiration rates. Beige cells have a gene expression pattern distinct from either white or brown fat and are preferentially sensitive to the polypeptide hormone irisin. Finally, we provide evidence that previously identified brown fat deposits in adult humans are composed of beige adipocytes. These data provide a foundation for studying this mammalian cell type with therapeutic potential. PAPERCLIP:
Asunto(s)
Adipocitos/clasificación , Adipocitos/metabolismo , Adipocitos Blancos/metabolismo , Tejido Adiposo Pardo/metabolismo , Animales , Separación Celular , Perfilación de la Expresión Génica , Humanos , Canales Iónicos/metabolismo , Ratones , Proteínas Mitocondriales/metabolismo , Proteína Desacopladora 1RESUMEN
The heart has the ability to grow in size in response to exercise, but little is known about the transcriptional mechanisms underlying physiological hypertrophy. Adult cardiomyocytes have also recently been proven to hold the potential for proliferation, a process that could be of great importance for regenerative medicine. Using a unique RT-PCR-based screen against all transcriptional components, we showed that C/EBPß was downregulated with exercise, whereas the expression of CITED4 was increased. Reduction of C/EBPß in vitro and in vivo resulted in a phenocopy of endurance exercise with cardiomyocyte hypertrophy and proliferation. This proliferation was mediated, at least in part, by the increased CITED4. Importantly, mice with reduced cardiac C/EBPß levels displayed substantial resistance to cardiac failure upon pressure overload. These data indicate that C/EBPß represses cardiomyocyte growth and proliferation in the adult mammalian heart and that reduction in C/EBPß is a central signal in physiologic hypertrophy and proliferation.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Corazón/fisiología , Condicionamiento Físico Animal , Animales , Proliferación Celular , Células Cultivadas , Embrión no Mamífero/metabolismo , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Miocardio/citología , Miocitos Cardíacos/metabolismo , Ratas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Pez Cebra/embriologíaRESUMEN
Exercise benefits a variety of organ systems in mammals, and some of the best-recognized effects of exercise on muscle are mediated by the transcriptional co-activator PPAR-γ co-activator-1 α (PGC1-α). Here we show in mouse that PGC1-α expression in muscle stimulates an increase in expression of FNDC5, a membrane protein that is cleaved and secreted as a newly identified hormone, irisin. Irisin acts on white adipose cells in culture and in vivo to stimulate UCP1 expression and a broad program of brown-fat-like development. Irisin is induced with exercise in mice and humans, and mildly increased irisin levels in the blood cause an increase in energy expenditure in mice with no changes in movement or food intake. This results in improvements in obesity and glucose homeostasis. Irisin could be therapeutic for human metabolic disease and other disorders that are improved with exercise.
Asunto(s)
Tejido Adiposo Pardo/citología , Tejido Adiposo Blanco/citología , Termogénesis , Transactivadores/metabolismo , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Tejido Adiposo Pardo/efectos de los fármacos , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Animales , Respiración de la Célula/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/genética , Metabolismo Energético/fisiología , Ejercicio Físico/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Hormonas/metabolismo , Humanos , Resistencia a la Insulina/fisiología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Canales Iónicos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Proteínas Mitocondriales/metabolismo , Modelos Animales , Células Musculares/metabolismo , Obesidad/sangre , Obesidad/inducido químicamente , Obesidad/prevención & control , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Condicionamiento Físico Animal/fisiología , Plasma/química , Grasa Subcutánea/citología , Grasa Subcutánea/efectos de los fármacos , Grasa Subcutánea/metabolismo , Termogénesis/efectos de los fármacos , Termogénesis/genética , Transactivadores/deficiencia , Transactivadores/genética , Factores de Transcripción , Proteína Desacopladora 1RESUMEN
Obesity induced in mice by high-fat feeding activates the protein kinase Cdk5 (cyclin-dependent kinase 5) in adipose tissues. This results in phosphorylation of the nuclear receptor PPARgamma (peroxisome proliferator-activated receptor gamma), a dominant regulator of adipogenesis and fat cell gene expression, at serine 273. This modification of PPARgamma does not alter its adipogenic capacity, but leads to dysregulation of a large number of genes whose expression is altered in obesity, including a reduction in the expression of the insulin-sensitizing adipokine, adiponectin. The phosphorylation of PPARgamma by Cdk5 is blocked by anti-diabetic PPARgamma ligands, such as rosiglitazone and MRL24. This inhibition works both in vivo and in vitro, and is completely independent of classical receptor transcriptional agonism. Similarly, inhibition of PPARgamma phosphorylation in obese patients by rosiglitazone is very tightly associated with the anti-diabetic effects of this drug. All these findings strongly suggest that Cdk5-mediated phosphorylation of PPARgamma may be involved in the pathogenesis of insulin-resistance, and present an opportunity for development of an improved generation of anti-diabetic drugs through PPARgamma.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Diabetes Mellitus Experimental/tratamiento farmacológico , Obesidad/metabolismo , PPAR gamma/metabolismo , Tiazolidinedionas/farmacología , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Tejido Adiposo/fisiopatología , Secuencia de Aminoácidos , Animales , Línea Celular , Quinasa 5 Dependiente de la Ciclina/genética , Quinasa 5 Dependiente de la Ciclina/metabolismo , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Grasas de la Dieta/farmacología , Humanos , Insulina/metabolismo , Ligandos , Ratones , Modelos Moleculares , Obesidad/inducido químicamente , Obesidad/complicaciones , Obesidad/fisiopatología , PPAR gamma/agonistas , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Conformación Proteica , Rosiglitazona , Tiazolidinedionas/uso terapéuticoRESUMEN
The accumulation of cytosolic lipid droplets in muscle and liver cells has been linked to the development of insulin resistance and type 2 diabetes. Such droplets are formed as small structures that increase in size through fusion, a process that is dependent on intact microtubules and the motor protein dynein. Approximately 15% of all droplets are involved in fusion processes at a given time. Here, we show that lipid droplets are associated with proteins involved in fusion processes in the cell: NSF (N-ethylmaleimide-sensitive-factor), alpha-SNAP (soluble NSF attachment protein) and the SNAREs (SNAP receptors), SNAP23 (synaptosomal-associated protein of 23 kDa), syntaxin-5 and VAMP4 (vesicle-associated membrane protein 4). Knockdown of the genes for SNAP23, syntaxin-5 or VAMP4, or microinjection of a dominant-negative mutant of alpha-SNAP, decreases the rate of fusion and the size of the lipid droplets. Thus, the SNARE system seems to have an important role in lipid droplet fusion. We also show that oleic acid treatment decreases the insulin sensitivity of heart muscle cells, and this sensitivity is completely restored by transfection with SNAP23. Thus, SNAP23 might be a link between insulin sensitivity and the inflow of fatty acids to the cell.
Asunto(s)
Citosol/metabolismo , Resistencia a la Insulina , Lípidos/fisiología , Fusión de Membrana , Ácido Oléico/farmacología , Proteínas SNARE/fisiología , Animales , Línea Celular , Ratones , Células 3T3 NIHRESUMEN
Comprehensive analysis of triacylglycerol (TAG) regioisomers is extremely challenging, with many variables that can influence the results. Previously, we reported a novel algorithmic method for resolving regioisomers of complex mixtures of TAGs. In the current study, the TAG Analyzer software and its mass spectrometric fragmentation model were further developed and validated for a much wider range of TAGs. To demonstrate the method, we performed for the first time a comprehensive analysis of TAG regioisomers of bovine milk fat, a very important and one of the most complex TAG mixtures in nature containing FAs ranging from short to long carbon chains. This analysis method forms a solid basis for further investigation of TAG regioisomer profiles in various natural fats and oils, potentially aiding in the development of new and healthier foods and nutraceuticals with targeted lipid structures.
Asunto(s)
Leche , Espectrometría de Masas en Tándem , Animales , Triglicéridos/química , Leche/química , Grasas/análisis , Programas InformáticosRESUMEN
BACKGROUND: Heart failure is a growing cause of morbidity and mortality. Cardiac phosphatidylinositol 3-kinase signaling promotes cardiomyocyte survival and function, but it is paradoxically activated in heart failure, suggesting that chronic activation of this pathway may become maladaptive. Here, we investigated the downstream phosphatidylinositol 3-kinase effector, serum- and glucocorticoid-regulated kinase-1 (SGK1), in heart failure and its complications. METHODS AND RESULTS: We found that cardiac SGK1 is activated in human and murine heart failure. We investigated the role of SGK1 in the heart by using cardiac-specific expression of constitutively active or dominant-negative SGK1. Cardiac-specific activation of SGK1 in mice increased mortality, cardiac dysfunction, and ventricular arrhythmias. The proarrhythmic effects of SGK1 were linked to biochemical and functional changes in the cardiac sodium channel and could be reversed by treatment with ranolazine, a blocker of the late sodium current. Conversely, cardiac-specific inhibition of SGK1 protected mice after hemodynamic stress from fibrosis, heart failure, and sodium channel alterations. CONCLUSIONS: SGK1 appears both necessary and sufficient for key features of adverse ventricular remodeling and may provide a novel therapeutic target in cardiac disease.
Asunto(s)
Cardiomiopatía Dilatada/enzimología , Insuficiencia Cardíaca/enzimología , Proteínas Inmediatas-Precoces/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Remodelación Ventricular/fisiología , Acetanilidas/uso terapéutico , Animales , Cardiomegalia Inducida por el Ejercicio , Secuencia de Consenso , Modelos Animales de Enfermedad , Electrocardiografía , Inducción Enzimática , Humanos , Hipertensión/complicaciones , Proteínas Inmediatas-Precoces/química , Proteínas Inmediatas-Precoces/deficiencia , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Canal de Sodio Activado por Voltaje NAV1.5/química , Canal de Sodio Activado por Voltaje NAV1.5/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.5/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Fosforilación , Piperazinas/uso terapéutico , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ranolazina , Bloqueadores de los Canales de Sodio/farmacología , Bloqueadores de los Canales de Sodio/uso terapéutico , Taquicardia Ventricular/enzimología , Taquicardia Ventricular/etiologíaRESUMEN
Exercise is known to prevent and treat metabolic diseases such as diabetes. However, the underlying mechanisms are not fully understood, and there is currently much focus on detailing such pathways. Traditionally, much emphasis has been placed on skeletal muscle; however, recently, nonmuscle organs such as adipose tissue have been highlighted in mediating protective actions after training. Moreover, novel paracrine- and endocrine-signaling molecules have been shown to trigger important responses in nonmuscle organs after exercise. This is exciting because, when administered exogenously, such signals have obvious therapeutic potential. In this review, the authors have described some general and historical aspects of training and disease protection. The authors have also highlighted some of the current knowledge on how exercise impacts nonmuscle organs.
Asunto(s)
Ejercicio Físico , Músculo Esquelético/metabolismo , Tejido Adiposo/metabolismo , Encéfalo/metabolismo , Encéfalo/fisiología , Humanos , Hígado/metabolismo , Hígado/fisiología , Músculo Esquelético/fisiología , Miocardio/metabolismoRESUMEN
Regioisomeric analysis of triacylglycerols (TAGs) in natural oils and fats is a highly challenging task in analytical chemistry. Here we present a software (TAG Analyzer) for automatic calculation of regioisomeric composition of TAGs based on the mass spectral data from recently reported ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method for analyzing TAG regioisomers. The software enables fast and accurate processing of complex product ion spectra containing structurally informative diacylglycerol [M+NH4-RCO2H-NH3]+ and fatty acid ketene [RCO]+ fragment ions. Compared to manual processing, the developed software offers higher throughput with faster calculation as well as more accurate interpretation of chromatographically overlapping isobaric TAGs. The software determines results by constructing a synthetic spectrum to match the measured fragment ion spectrum, and by reporting the optimal concentrations of TAGs used to create the synthetic spectrum. This type of calculation is often extremely challenging for manual interpretation of the fragment ion spectra of isobaric TAGs with shared fragments, hence the need for automated data processing. The developed software was validated by analyzing a wide range of mixtures of regiopure TAG reference compounds of known composition and a commercial olive oil sample. Additionally, the method was also applied for regiospecific analysis of TAGs in human milk as an example of natural fats and oils with a highly complex TAG profile. The results indicate that the software is capable of resolving regioisomeric composition of natural TAGs even of the most complex composition. This novel calculation software combined with our existing UHPLC-ESI-MS/MS method form a highly efficient tool for regioisomeric analysis of TAGs in natural fats and oils.
Asunto(s)
Aceites de Plantas , Espectrometría de Masas en Tándem , Cromatografía Líquida de Alta Presión/métodos , Grasas , Humanos , Aceites de Plantas/química , Programas Informáticos , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Triglicéridos/análisisRESUMEN
Neutral lipids are stored in the cytosol in so-called lipid droplets. These are dynamic organelles with neutral lipids as the core surrounded by a monolayer of amphipathic lipids (phospholipids and cholesterol) and specific proteins (PAT proteins and proteins involved in the turnover of lipids and in the formation and trafficking of the droplets). Lipid droplets are formed at microsomal membranes as primordial droplets with a diameter of 0.1-0.4 microm and increase in size by fusion. In this article, we review the assembly and fusion of lipid droplets, and the processes involved in the secretion of triglycerides. Triglycerides are secreted from cells by two principally different processes. In the mammary gland, lipid droplets interact with specific regions of the plasma membrane and bud off with an envelope consisting of the membrane, to form milk globules. In the liver and intestine, very low-density lipoproteins (VLDL) and chylomicrons are secreted by using the secretory pathway of the cell. Finally, we briefly review the importance of lipid droplets in the development of insulin resistance and atherosclerosis.
Asunto(s)
Orgánulos/metabolismo , Triglicéridos/metabolismo , Aciltransferasas/metabolismo , Animales , Aterosclerosis/metabolismo , Quilomicrones/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucolípidos/metabolismo , Glicoproteínas/metabolismo , Humanos , Resistencia a la Insulina , Gotas Lipídicas , Lipoproteínas VLDL/metabolismo , Fusión de Membrana , Microsomas/metabolismo , Tamaño de los Orgánulos , Transporte de ProteínasRESUMEN
The assembly of lipid droplets is dependent on PtdIns(4,5)P(2) that activates PLD(1) (phospholipase D(1)), which is important for the assembly process. ERK2 (extracellular-signal-regulated kinase 2) phosphorylates the motor protein dynein and sorts it to lipid droplets, allowing them to be transported on microtubules. Lipid droplets grow in size by fusion, which is dependent on dynein and the transfer on microtubules, and is catalysed by the SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins SNAP-23 (23 kDa synaptosome-associated protein), syntaxin-5 and VAMP-4 (vesicle-associated protein 4). SNAP-23 is also involved in the insulin-dependent translocation of the glucose transporter GLUT4 to the plasma membrane. Fatty acids induce a missorting of SNAP-23, from the plasma membrane to the interior of the cell, resulting in cellular insulin resistance that can be overcome by increasing the levels of SNAP-23. The same missorting of SNAP-23 occurs in vivo in skeletal-muscle biopsies from patients with T2D (Type 2 diabetes). Moreover, there was a linear relation between the amount of SNAP-23 in the plasma membrane from human skeletal-muscles biopsies and the systemic insulin-sensitivity. Syntaxin-5 is low in T2D patients, which leads to a decrease in the insulin-dependent phosphorylation of Akt (also known as protein kinase B). Thus both SNAP-23 and syntaxin-5 are highly involved in the development of insulin resistance.
Asunto(s)
Ácidos Grasos/metabolismo , Resistencia a la Insulina , Metabolismo de los Lípidos , Orgánulos , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Ácidos Grasos/química , Humanos , Insulina/metabolismo , Microsomas/metabolismo , Microsomas/ultraestructura , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Orgánulos/química , Orgánulos/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/genéticaRESUMEN
Triglyceride-containing lipid droplets (LD) are dynamic organelles stored on demand in all cells. These droplets grow through a fusion process mediated by SNARE proteins, including SNAP23. The droplets have also been shown to be highly motile and interact with other cell organelles, including peroxisomes and the endoplasmic reticulum. We have used electron and confocal microscopy to demonstrate that LD form complexes with mitochondria in NIH 3T3 fibroblasts. Using an in vitro system of purified LD and mitochondria, we also show the formation of the LD-mitochondria complex, in which cytosolic factors are involved. Moreover, the presence of LD markers in mitochondria isolated by subcellular fractionations is demonstrated. Finally, ablation of SNAP23 using siRNA reduced complex formation and beta oxidation, which suggests that the LD-mitochondria complex is functional in the cell.
Asunto(s)
Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Triglicéridos/metabolismo , Animales , Técnicas de Silenciamiento del Gen , Proteínas de la Membrana/agonistas , Ratones , Microscopía Electrónica de Transmisión , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Células 3T3 NIH , Ácido Oléico/farmacología , Oxidación-Reducción , Perilipina-2 , Prohibitinas , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/genética , ARN Interferente Pequeño/genética , Proteínas Represoras/metabolismoRESUMEN
OBJECTIVE: We investigated the role of adipocyte differentiation-related protein (ADRP) in triglyceride turnover and in the secretion of very low-density lipoprotein (VLDL) from McA-RH7777 cells and primary rat hepatocytes. METHODS AND RESULTS: An increase in the expression of ADRP increased triglyceride accumulation in cytosolic lipid droplets and prevented the incorporation of fatty acids into secretable triglycerides, thereby reducing the secretion of triglycerides as well as of apolipoprotein B-100 (apoB-100) and apoB-48 VLDL. The ability of ADRP to block the secretion of apoB-100 VLDL1 decreased with increasing quantities of fatty acids in the medium, indicating a saturable process and emphasizing the importance of sequestering of fatty acids for the effect of ADRP on VLDL secretion. Knockdown (small interfering RNA) of ADRP decreased the pool of cytosolic lipid droplets but increased only the secretion of apoB-48 VLDL1. Additionally, there was an increased flow of fatty acids into beta-oxidation. CONCLUSIONS: ADRP is essential for the accumulation of triglycerides in cytosolic lipid droplets. An increase in ADRP prevents the formation of VLDL by diverting fatty acids from the VLDL assembly pathway into cytosolic triglycerides, whereas a decrease of the protein increases the sorting of fatty acids to beta-oxidation and promotes the secretion of apoB-48 VLDL1.
Asunto(s)
Citosol/metabolismo , Ácidos Grasos/metabolismo , Lipoproteínas VLDL/antagonistas & inhibidores , Proteínas de la Membrana/fisiología , Triglicéridos/metabolismo , Animales , Apolipoproteína B-48 , Apolipoproteínas B/metabolismo , Línea Celular Tumoral , Técnicas de Transferencia de Gen , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Lipoproteínas VLDL/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/farmacología , Oxidación-Reducción/efectos de los fármacos , Perilipina-2 , ARN Interferente Pequeño/farmacología , RatasRESUMEN
OBJECTIVE: Atherosclerotic lesions have regions that are hypoxic. Because the lesion contains macrophages that are loaded with lipid, we investigated whether hypoxia can influence the accumulation of lipids in these cells. METHODS AND RESULTS: Exposure of human macrophages to hypoxia for 24 hours resulted in an increased formation of cytosolic lipid droplets and an increased accumulation of triglycerides. Exposure of the macrophages to oxidized low-density lipoprotein (oxLDL) increased the accumulation of cytosolic lipid droplets because of an increase in cellular cholesterol esters. The accumulation of lipid droplets in oxLDL-treated cells was further increased after hypoxia, caused by an increased level of triglycerides. Expression analyses combined with immunoblot or RT-PCR demonstrated that hypoxia increased the expression of several genes that could promote the accumulation of lipid droplets. Hypoxia increased the mRNA and protein levels of adipocyte differentiation-related protein (ADRP). It is well known that an increased expression of ADRP increases the formation of lipid droplets. Hypoxia decreased the expression of enzymes involved in beta-oxidation (acyl-coenzyme A synthetase and acyl-coenzyme A dehydrogenase) and increased the expression of stearoyl-coenzyme A desaturase, an important enzyme in the fatty acid biosynthesis. Moreover, exposure to hypoxia decreased the rate of beta-oxidation, whereas the accumulation of triglycerides increased. CONCLUSIONS: The results demonstrate that exposure of human macrophages to hypoxia causes an accumulation of triglyceride-containing cytosolic lipid droplets. This indicates that the hypoxia present in atherosclerotic lesions can contribute to the formation of the lipid-loaded macrophages that characterize the lesion and to the accumulation of triglycerides in such lesions.
Asunto(s)
Células Espumosas/metabolismo , Células Espumosas/patología , Hipoxia/metabolismo , Hipoxia/patología , Metabolismo de los Lípidos , Macrófagos/metabolismo , Triglicéridos/metabolismo , Acil-CoA Deshidrogenasa/antagonistas & inhibidores , Células Cultivadas , Coenzima A Ligasas/antagonistas & inhibidores , Citosol/metabolismo , Humanos , Immunoblotting , Metabolismo de los Lípidos/efectos de los fármacos , Lipoproteínas LDL/farmacología , Macrófagos/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Perilipina-2 , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estearoil-CoA Desaturasa/metabolismo , Factores de TiempoRESUMEN
A highly sensitive mass spectrometric (MS) method was developed and validated to analyze ratios of regioisomeric triacylglycerols (TAGs) in fats and oils. UPLC resolution of lithiated TAGs followed by daughter scan MS/MS of positive ions revealed several indicative ions for quantitative analysis. Reference TAGs containing C14-C20 fatty acids (FAs) showed good linear response. Analysis of Finnish and Chinese pooled human milk samples revealed hundreds of regioisomeric TAGs. At least 64mol% of the TAGs were quantified with relative standard deviation <17%. When present in the same TAG molecule together with C18 FAs, palmitic acid was typically in the sn-2 position. When together with FAs 10:0, 12:0, 14:0, 20:1 and 20:2, the sn-2 preference of 16:0 was less clear. Oleic acid occupied typically the sn-1/sn-3 positions but when together with FAs 20:1, 20:2, 18:2, 14:1, 12:0 or 10:0 the positioning of 18:1 did not follow these rules.
Asunto(s)
Espectrometría de Masas en Tándem , Algoritmos , Humanos , Espectrometría de Masas , Leche Humana , Aceites de Plantas , Espectrometría de Masa por Ionización de Electrospray , TriglicéridosRESUMEN
OBJECTIVE: Adipocyte differentiation-related protein (ADRP)-containing lipid droplets have an essential role in the development of insulin resistance and atherosclerosis. Such droplets form in a cell-free system with a diameter of 0.1 to 0.4 microm, while the droplets present in cells vary in size, from small to very large, suggesting that the droplets can increase in size after being assembled. We have addressed this possibility. METHODS AND RESULTS: Experiments in NIH 3T3 cells demonstrated that the lipid droplets could increase in size independently of triglyceride biosynthesis. NIH 3T3 cells were either microinjected with ADRP-GFP (green fluorescent protein) or stained with Nile Red and followed by confocal microscopy and time-lapse recordings. The results showed that lipid droplets formed complexes with each other, with a volume equal to the sum of the merging particles. The formation of complexes could be inhibited by the nocodazole-induced depolymerization of the microtubules; thus, the process is dependent on microtubules. The presence of dynein on ADRP-containing droplets supports a role for this motor protein. CONCLUSIONS: Lipid droplets can grow after they have been assembled. This increase in size is independent of triglyceride biosynthesis and involves formation of complexes, which requires intact microtubules.
Asunto(s)
Metabolismo de los Lípidos , Microtúbulos/metabolismo , Ácido Oléico/farmacocinética , Animales , Antineoplásicos/farmacología , Aterosclerosis/metabolismo , Citosol/metabolismo , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes/farmacología , Proteínas de la Membrana/farmacología , Ratones , Microtúbulos/efectos de los fármacos , Proteínas Motoras Moleculares/metabolismo , Células 3T3 NIH , Nocodazol/farmacología , Ácido Oléico/química , Oxazinas , Tamaño de la Partícula , Perilipina-2 , Triglicéridos/biosíntesisRESUMEN
FNDC4 is a secreted factor sharing high homology with the exercise-associated myokine irisin (FNDC5). Here we report that Fndc4 is robustly upregulated in several mouse models of inflammation as well as in human inflammatory conditions. Specifically, FNDC4 levels are increased locally at inflamed sites of the intestine of inflammatory bowel disease patients. Interestingly, administration of recombinant FNDC4 in the mouse model of induced colitis markedly reduces disease severity compared with mice injected with a control protein. Conversely, mice lacking Fndc4 develop more severe colitis. Analysis of binding of FNDC4 to different immune cell types reveals strong and specific binding to macrophages and monocytes. FNDC4 treatment of bone marrow-derived macrophages in vitro results in reduced phagocytosis, increased cell survival and reduced proinflammatory chemokine expression. Hence, treatment with FNDC4 results in a state of dampened macrophage activity, while enhancing their survival. Thus, we have characterized FNDC4 as a factor with direct therapeutic potential in inflammatory bowel disease and possibly other inflammatory diseases.
Asunto(s)
Antiinflamatorios/metabolismo , Colitis/metabolismo , Macrófagos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Colitis/genética , Colitis/patología , Sulfato de Dextran , Progresión de la Enfermedad , Regulación de la Expresión Génica , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Fagocitosis/efectos de los fármacos , Proteínas/química , Proteínas/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Exercise induces physiological cardiac growth and protects the heart against pathological remodeling. Recent work suggests exercise also enhances the heart's capacity for repair, which could be important for regenerative therapies. While microRNAs are important in certain cardiac pathologies, less is known about their functional roles in exercise-induced cardiac phenotypes. We profiled cardiac microRNA expression in two distinct models of exercise and found microRNA-222 (miR-222) was upregulated in both. Downstream miR-222 targets modulating cardiomyocyte phenotypes were identified, including HIPK1 and HMBOX1. Inhibition of miR-222 in vivo completely blocked cardiac and cardiomyocyte growth in response to exercise while reducing markers of cardiomyocyte proliferation. Importantly, mice with inducible cardiomyocyte miR-222 expression were resistant to adverse cardiac remodeling and dysfunction after ischemic injury. These studies implicate miR-222 as necessary for exercise-induced cardiomyocyte growth and proliferation in the adult mammalian heart and show that it is sufficient to protect the heart against adverse remodeling.