RESUMEN
The calculation of the 13 C and 15 N NMR chemical shifts by a combined molecular mechanics (Pcmodel 9.1/MMFF94) and ab initio (GIAO (B3LYP/DFT, 6-31 + G(d)) procedure is used to investigate the conformations of a variety of alkyl substituted anilines. The 13 C shifts are obtained from the GIAO isotropic shielding (Ciso) with separate references for sp3 and sp2 carbons (δc = δref - Ciso). The 15 N shifts are obtained similarly from the GIAO isotropic shielding (Niso) with reference to the 15 N chemical shift of aniline. Comparison of the observed and calculated shifts provides information on the molecular conformations. Aniline and the 2,6-dialkylanilines exist with a rapidly inverting symmetric pyramidal nitrogen atom. The 2-alkylanilines have similar conformations with the NH2 group tilted away from the 2-alkyl substituent. The N,N-dialkylanilines show more varied conformations. N,N-dimethylaniline has a similar structure to aniline, but N-ethyl, N-methylaniline, N,N-diethylaniline, and N,N-diisopropylaniline are conformationally mobile with two rapidly interconverting conformers. In contrast, the anilines substituted at C2 and the nitrogen atom exist as one conformer where the steric interaction between the C2 substituent and the N substituent determines the conformation. In 2-methyl-N-methylaniline, the nitrogen atom is pyramidal as usual with the N-methyl opposite to the 2-methyl, but in 2-methyl-N,N-dimethyl aniline, the NMe2 group is now almost orthogonal to the phenyl plane. This is also the case with 2-methyl-N,N-diethylaniline and 2,6-diisopropyl-N,N-dimethylaniline. The comparison of the observed and calculated 15 N chemical shifts confirms the above findings, in particular the pyramidal conformation of aniline and the above observations with respect to the conformations of the N,N-dialkylanilines.
Asunto(s)
Compuestos de Anilina/química , Teoría Cuántica , Isótopos de Carbono , Espectroscopía de Resonancia Magnética/normas , Conformación Molecular , Isótopos de NitrógenoRESUMEN
Telomere dysfunction activates p53-mediated cellular growth arrest, senescence and apoptosis to drive progressive atrophy and functional decline in high-turnover tissues. The broader adverse impact of telomere dysfunction across many tissues including more quiescent systems prompted transcriptomic network analyses to identify common mechanisms operative in haematopoietic stem cells, heart and liver. These unbiased studies revealed profound repression of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha and beta (PGC-1α and PGC-1ß, also known as Ppargc1a and Ppargc1b, respectively) and the downstream network in mice null for either telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) genes. Consistent with PGCs as master regulators of mitochondrial physiology and metabolism, telomere dysfunction is associated with impaired mitochondrial biogenesis and function, decreased gluconeogenesis, cardiomyopathy, and increased reactive oxygen species. In the setting of telomere dysfunction, enforced Tert or PGC-1α expression or germline deletion of p53 (also known as Trp53) substantially restores PGC network expression, mitochondrial respiration, cardiac function and gluconeogenesis. We demonstrate that telomere dysfunction activates p53 which in turn binds and represses PGC-1α and PGC-1ß promoters, thereby forging a direct link between telomere and mitochondrial biology. We propose that this telomere-p53-PGC axis contributes to organ and metabolic failure and to diminishing organismal fitness in the setting of telomere dysfunction.
Asunto(s)
Mitocondrias/metabolismo , Mitocondrias/patología , Telómero/metabolismo , Telómero/patología , Adenosina Trifosfato/biosíntesis , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Cardiomiopatías/inducido químicamente , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Cardiomiopatías/fisiopatología , Proliferación Celular , ADN Mitocondrial/análisis , Doxorrubicina/toxicidad , Gluconeogénesis , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Hígado/citología , Hígado/metabolismo , Ratones , Miocardio/citología , Miocardio/metabolismo , ARN/genética , Especies Reactivas de Oxígeno/metabolismo , Telomerasa/deficiencia , Telomerasa/genética , Telómero/enzimología , Telómero/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
RATIONALE: Mitochondria, although required for cellular ATP production, are also known to have other important functions that may include modulating cellular responses to environmental stimuli. However, the mechanisms whereby mitochondria impact cellular phenotype are not yet clear. OBJECTIVE: To determine how mitochondria impact endothelial cell function. METHODS AND RESULTS: We report here that stimuli for endothelial cell proliferation evoke strong upregulation of mitochondrial uncoupling protein 2 (UCP2). Analysis in silico indicated increased UCP2 expression is common in highly proliferative cell types, including cancer cells. Upregulation of UCP2 was critical for controlling mitochondrial membrane potential (Δψ) and superoxide production. In the absence of UCP2, endothelial growth stimulation provoked mitochondrial network fragmentation and premature senescence via a mechanism involving superoxide-mediated p53 activation. Mitochondrial network fragmentation was both necessary and sufficient for the impact of UCP2 on endothelial cell phenotype. CONCLUSIONS: These data identify a novel mechanism whereby mitochondria preserve normal network integrity and impact cell phenotype via dynamic regulation of UCP2.
Asunto(s)
Células Endoteliales/metabolismo , Canales Iónicos/metabolismo , Dinámicas Mitocondriales , Proteínas Mitocondriales/metabolismo , Fenotipo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Aorta/citología , Bovinos , Proliferación Celular , Senescencia Celular , Células Endoteliales/citología , Canales Iónicos/genética , Pulmón/citología , Potencial de la Membrana Mitocondrial , Ratones , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Superóxidos/metabolismo , Proteína Desacopladora 2 , Regulación hacia ArribaRESUMEN
Hepatic gluconeogenesis is required for maintaining blood glucose homeostasis; yet, in diabetes mellitus, this process is unrestrained and is a major contributor to fasting hyperglycemia. To date, the impacts of chromatin modifying enzymes and chromatin landscape on gluconeogenesis are poorly understood. Through catalyzing the removal of methyl groups from specific lysine residues in the histone tail, histone demethylases modulate chromatin structure and, hence, gene expression. Here we perform an RNA interference screen against the known histone demethylases and identify a histone H3 lysine 36 (H3K36) demethylase, Jhdm1a, as a key negative regulator of gluconeogenic gene expression. In vivo, silencing of Jhdm1a promotes liver glucose synthesis, while its exogenous expression reduces blood glucose level. Importantly, the regulation of gluconeogenesis by Jhdm1a requires its demethylation activity. Mechanistically, we find that Jhdm1a regulates the expression of a major gluconeogenic regulator, C/EBPα. This is achieved, at least in part, by its USF1-dependent association with the C/EBPα promoter and its subsequent demethylation of dimethylated H3K36 on the C/EBPα locus. Our work provides compelling evidence that links histone demethylation to transcriptional regulation of gluconeogenesis and has important implications for the treatment of diabetes.
Asunto(s)
Glucemia , Gluconeogénesis , Histona Demetilasas con Dominio de Jumonji , Hígado/metabolismo , Animales , Glucemia/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Células Cultivadas , Regulación de la Expresión Génica , Gluconeogénesis/genética , Glucosa-6-Fosfatasa/metabolismo , Hepatocitos/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/genética , Ratas , Factores Estimuladores hacia 5'/metabolismoRESUMEN
Ischaemia of the heart, brain and limbs is a leading cause of morbidity and mortality worldwide. Hypoxia stimulates the secretion of vascular endothelial growth factor (VEGF) and other angiogenic factors, leading to neovascularization and protection against ischaemic injury. Here we show that the transcriptional coactivator PGC-1alpha (peroxisome-proliferator-activated receptor-gamma coactivator-1alpha), a potent metabolic sensor and regulator, is induced by a lack of nutrients and oxygen, and PGC-1alpha powerfully regulates VEGF expression and angiogenesis in cultured muscle cells and skeletal muscle in vivo. PGC-1alpha-/- mice show a striking failure to reconstitute blood flow in a normal manner to the limb after an ischaemic insult, whereas transgenic expression of PGC-1alpha in skeletal muscle is protective. Surprisingly, the induction of VEGF by PGC-1alpha does not involve the canonical hypoxia response pathway and hypoxia inducible factor (HIF). Instead, PGC-1alpha coactivates the orphan nuclear receptor ERR-alpha (oestrogen-related receptor-alpha) on conserved binding sites found in the promoter and in a cluster within the first intron of the VEGF gene. Thus, PGC-1alpha and ERR-alpha, major regulators of mitochondrial function in response to exercise and other stimuli, also control a novel angiogenic pathway that delivers needed oxygen and substrates. PGC-1alpha may provide a novel therapeutic target for treating ischaemic diseases.
Asunto(s)
Isquemia/metabolismo , Neovascularización Fisiológica , Transactivadores/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Hipoxia de la Célula , Células Cultivadas , Regulación de la Expresión Génica , Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Ratones Transgénicos , Músculo Esquelético/metabolismo , Oxígeno/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Receptores de Estrógenos/metabolismo , Transactivadores/deficiencia , Transactivadores/genética , Factores de Transcripción , Transgenes/genética , Receptor Relacionado con Estrógeno ERRalfaRESUMEN
Impaired oxidative phosphorylation (OXPHOS) is implicated in several metabolic disorders. Even though mitochondrial DNA encodes several subunits critical for OXPHOS, the metabolic consequence of activating mitochondrial transcription remains unclear. We show here that LRP130, a protein involved in Leigh syndrome, increases hepatic ß-fatty acid oxidation. Using convergent genetic and biochemical approaches, we demonstrate LRP130 complexes with the mitochondrial RNA polymerase to activate mitochondrial transcription. Activation of mitochondrial transcription is associated with increased OXPHOS activity, increased supercomplexes, and denser cristae, independent of mitochondrial biogenesis. Consistent with increased oxidative phosphorylation, ATP levels are increased in both cells and mouse liver, whereas coupled respiration is increased in cells. We propose activation of mitochondrial transcription remodels mitochondria and enhances oxidative metabolism.
Asunto(s)
Ácidos Grasos/metabolismo , Mitocondrias Hepáticas/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Neoplasias/metabolismo , Fosforilación Oxidativa , Animales , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Ácidos Grasos/genética , Células Hep G2 , Humanos , Enfermedad de Leigh/genética , Enfermedad de Leigh/metabolismo , Ratones , Mitocondrias Hepáticas/genética , Proteínas Mitocondriales/genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas de Neoplasias/genética , Oxidación-Reducción , Consumo de Oxígeno/fisiología , Transcripción Genética/fisiologíaRESUMEN
TRPM2 Ca(2+)-permeable cation channel is widely expressed and activated by markers of cellular stress. Since inflammation and stress play a major role in insulin resistance, we examined the role of TRPM2 Ca(2+) channel in glucose metabolism. A 2-h hyperinsulinemic euglycemic clamp was performed in TRPM2-deficient (KO) and wild-type mice to assess insulin sensitivity. To examine the effects of diet-induced obesity, mice were fed a high-fat diet for 4-10 mo, and metabolic cage and clamp studies were conducted in conscious mice. TRPM2-KO mice were more insulin sensitive partly because of increased glucose metabolism in peripheral organs. After 4 mo of high-fat feeding, TRPM2-KO mice were resistant to diet-induced obesity, and this was associated with increased energy expenditure and elevated expressions of PGC-1α, PGC-1ß, PPARα, ERRα, TFAM, and MCAD in white adipose tissue. Hyperinsulinemic euglycemic clamps showed that TRPM2-KO mice were more insulin sensitive, with increased Akt and GSK-3ß phosphorylation in heart. Obesity-mediated inflammation in adipose tissue and liver was attenuated in TRPM2-KO mice. Overall, TRPM2 deletion protected mice from developing diet-induced obesity and insulin resistance. Our findings identify a novel role of TRPM2 Ca(2+) channel in the regulation of energy expenditure, inflammation, and insulin resistance.
Asunto(s)
Metabolismo Energético/fisiología , Glucosa/metabolismo , Canales Catiónicos TRPM/fisiología , Animales , Western Blotting , Composición Corporal/fisiología , Peso Corporal/fisiología , Calmodulina/metabolismo , Calorimetría Indirecta , Grasas de la Dieta/farmacología , Técnica de Clampeo de la Glucosa , Prueba de Tolerancia a la Glucosa , Inmunoprecipitación , Inflamación/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Resistencia a la Insulina/fisiología , Ratones , Ratones Noqueados , Miocardio/enzimología , Miocardio/metabolismo , Consumo de Oxígeno/fisiología , Fosforilación , ARN/biosíntesis , ARN/genética , Superóxido Dismutasa/metabolismoRESUMEN
BACKGROUND & AIMS: Mechanisms underlying synergistic liver injury caused by alcohol and obesity are not clear. We have produced a mouse model of synergistic steatohepatitis by recapitulating the natural history of the synergism seen in patients for mechanistic studies. METHODS: Moderate obesity was induced in mice by 170% overnutrition in calories using intragastric overfeeding of high fat diet. Alcohol (low or high dose) was then co-administrated to determine its effects. RESULTS: Moderate obesity plus alcohol intake causes synergistic steatohepatitis in an alcohol dose-dependent manner. A heightened synergism is observed when a high alcohol dose (32g/kg/d) is used, resulting in plasma ALT reaching 392±28U/L, severe steatohepatitis with pericellular fibrosis, marked M1 macrophage activation, a 40-fold induction of iNos, and intensified nitrosative stress in the liver. Hepatic expression of genes for mitochondrial biogenesis and metabolism are significantly downregulated, and hepatic ATP level is decreased. Synergistic ER stress evident by elevated XBP-1, GRP78 and CHOP is accompanied by hyperhomocysteinemia. Despite increased caspase 3/7 cleavage, their activities are decreased in a redox-dependent manner. Neither increased PARP cleavage nor TUNEL positive hepatocytes are found, suggesting a shift of apoptosis to necrosis. Surprisingly, the synergism mice have increased plasma adiponectin and hepatic p-AMPK, but adiponectin resistance is shown downstream of p-AMPK. CONCLUSIONS: Nitrosative stress mediated by M1 macrophage activation, adiponectin resistance, and accentuated ER and mitochondrial stress underlie potential mechanisms for synergistic steatohepatitis caused by moderate obesity and alcohol.
Asunto(s)
Etanol/farmacología , Hígado Graso/metabolismo , Expresión Génica/efectos de los fármacos , Hígado/metabolismo , Mitocondrias/genética , Obesidad/complicaciones , Transducción de Señal , Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Trifosfato/metabolismo , Adiponectina/metabolismo , Tejido Adiposo Blanco/metabolismo , Alanina Transaminasa/sangre , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Arginasa/genética , Arginasa/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Hígado Graso/etiología , Hígado Graso/genética , Hígado Graso/patología , Interleucina-10/genética , Interleucina-10/metabolismo , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico Sintasa de Tipo I/metabolismo , Obesidad/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoAsunto(s)
Metilación de ADN/fisiología , Endotelio Vascular/fisiología , Epigénesis Genética/fisiología , Neovascularización Fisiológica/fisiología , Animales , Supervivencia Celular/fisiología , Proteínas de Unión al ADN/fisiología , Endotelio Vascular/citología , Miembro Posterior/irrigación sanguínea , Humanos , Isquemia/prevención & control , Ratones , Modelos AnimalesRESUMEN
Mitochondrial respiration plays a crucial role in determining the metabolic state of brown adipose tissue (BAT), due to its direct roles in thermogenesis, as well as through additional mechanisms. Here, we show that respiration-dependent retrograde signaling from mitochondria to nucleus contributes to genetic and metabolic reprogramming of BAT. In mouse BAT, ablation of LRPPRC (LRP130), a potent regulator of mitochondrial transcription and respiratory capacity, triggers down-regulation of thermogenic genes, promoting a storage phenotype in BAT. This retrograde regulation functions by inhibiting the recruitment of PPARγ to the regulatory elements of thermogenic genes. Reducing cytosolic Ca2+ reverses the attenuation of thermogenic genes in brown adipocytes with impaired respiratory capacity, while induction of cytosolic Ca2+ is sufficient to attenuate thermogenic gene expression, indicating that cytosolic Ca2+ mediates mitochondria-nucleus crosstalk. Our findings suggest respiratory capacity governs thermogenic gene expression and BAT function via mitochondria-nucleus communication, which in turn leads to either a thermogenic or storage mode.
Asunto(s)
Respiración de la Célula , Regulación de la Expresión Génica , Mitocondrias/genética , Mitocondrias/metabolismo , Transducción de Señal , Termogénesis/genética , Tejido Adiposo Pardo/metabolismo , Animales , Calcio/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/ultraestructura , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , PPAR gamma/genética , PPAR gamma/metabolismo , Regiones Promotoras GenéticasRESUMEN
Uncoupling protein 1 (UCP1) is highly expressed in brown adipose tissue, where it generates heat by uncoupling electron transport from ATP production. UCP1 is also found outside classical brown adipose tissue depots, in adipocytes that are termed 'brite' (brown-in-white) or 'beige'. In humans, the presence of brite or beige (brite/beige) adipocytes is correlated with a lean, metabolically healthy phenotype, but whether a causal relationship exists is not clear. Here we report that human brite/beige adipocyte progenitors proliferate in response to pro-angiogenic factors, in association with expanding capillary networks. Adipocytes formed from these progenitors transform in response to adenylate cyclase activation from being UCP1 negative to being UCP1 positive, which is a defining feature of the beige/brite phenotype, while displaying uncoupled respiration. When implanted into normal chow-fed, or into high-fat diet (HFD)-fed, glucose-intolerant NOD-scid IL2rg(null) (NSG) mice, brite/beige adipocytes activated in vitro enhance systemic glucose tolerance. These adipocytes express neuroendocrine and secreted factors, including the pro-protein convertase PCSK1, which is strongly associated with human obesity. Pro-angiogenic conditions therefore drive the proliferation of human beige/brite adipocyte progenitors, and activated beige/brite adipocytes can affect systemic glucose homeostasis, potentially through a neuroendocrine mechanism.
Asunto(s)
Adipocitos/metabolismo , Glucemia/metabolismo , Intolerancia a la Glucosa/metabolismo , Neovascularización Fisiológica , Consumo de Oxígeno , ARN Mensajero/metabolismo , Adipocitos/trasplante , Adipocitos Marrones/metabolismo , Adipocitos Marrones/trasplante , Adipocitos Blancos/metabolismo , Adipocitos Blancos/trasplante , Adulto , Anciano , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Capilares , Trasplante de Células , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dieta Alta en Grasa , Encefalinas/genética , Encefalinas/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Técnica de Clampeo de la Glucosa , Prueba de Tolerancia a la Glucosa , Homeostasis , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Interleucina-33/genética , Interleucina-33/metabolismo , Yoduro Peroxidasa/genética , Yoduro Peroxidasa/metabolismo , Canales Iónicos/genética , Canales Iónicos/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Obesidad/metabolismo , Reacción en Cadena de la Polimerasa , Proproteína Convertasa 1/genética , Proproteína Convertasa 1/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteína Desacopladora 1 , Yodotironina Deyodinasa Tipo IIRESUMEN
Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders. In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via ß-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying ß-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of ß-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.
Asunto(s)
Ácidos Grasos/metabolismo , Hepatocitos/metabolismo , Animales , Técnicas de Cultivo de Célula/métodos , Separación Celular/métodos , Hepatocitos/citología , Metabolismo de los Lípidos , Lipogénesis , Hígado/citología , Hígado/metabolismo , Ratones , Oxidación-Reducción , Palmitatos/metabolismo , Radiofármacos/química , Radiofármacos/metabolismo , Tritio/químicaRESUMEN
In murine and human brown adipose tissue (BAT), mitochondria are powerful generators of heat that safely metabolize fat, a feature that has great promise in the fight against obesity and diabetes. Recent studies suggest that the actions of mitochondria extend beyond their conventional role as generators of heat. There is mounting evidence that impaired mitochondrial respiratory capacity is accompanied by attenuated expression of Ucp1 and other BAT-selective genes, implying that mitochondria exert transcriptional control over the brown fat gene program. In this review, we discuss the current understanding of brown fat mitochondria, their potential role in transcriptional control of the brown fat gene program, and potential strategies to treat obesity in humans by leveraging thermogenesis in brown adipocytes.
RESUMEN
OXPHOS is believed to play an important role in non-alcoholic fatty liver disease (NAFLD), however, precise mechanisms whereby OXPHOS influences lipid homeostasis are incompletely understood. We previously reported that ectopic expression of LRPPRC, a protein that increases cristae density and OXPHOS, promoted fatty acid oxidation in cultured primary hepatocytes. To determine the biological significance of that observation and define underlying mechanisms, we have ectopically expressed LRPPRC in mouse liver in the setting of NAFLD. Interestingly, ectopic expression of LRPPRC in mouse liver completely interdicted NAFLD, including inflammation. Consistent with mitigation of NAFLD, two markers of hepatic insulin resistance--ROS and PKCε activity--were both modestly reduced. As reported by others, improvement of NAFLD was associated with improved whole-body insulin sensitivity. Regarding hepatic lipid homeostasis, the ratio of NAD+ to NADH was dramatically increased in mouse liver replete with LRPPRC. Pharmacological activators and inhibitors of the cellular respiration respectively increased and decreased the [NAD+]/[NADH] ratio, indicating respiration-mediated control of the [NAD+]/[NADH] ratio. Supporting a prominent role for NAD+, increasing the concentration of NAD+ stimulated complete oxidation of fatty acids. Importantly, NAD+ rescued impaired fatty acid oxidation in hepatocytes deficient for either OXPHOS or SIRT3. These data are consistent with a model whereby augmented hepatic OXPHOS increases NAD+, which in turn promotes complete oxidation of fatty acids and protects against NAFLD.
Asunto(s)
Ácidos Grasos/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Mitocondrias/metabolismo , Proteínas de Neoplasias/genética , Enfermedad del Hígado Graso no Alcohólico/genética , Animales , Dieta Alta en Grasa , Regulación de la Expresión Génica , Hepatocitos/patología , Humanos , Resistencia a la Insulina , Metabolismo de los Lípidos/genética , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/genética , Mitocondrias/patología , NAD/metabolismo , Proteínas de Neoplasias/metabolismo , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Oxidación-Reducción , Fosforilación Oxidativa , Cultivo Primario de Células , Proteína Quinasa C-epsilon/genética , Proteína Quinasa C-epsilon/metabolismo , Transducción de Señal , Sirtuina 3/deficiencia , Sirtuina 3/genéticaRESUMEN
Signalling pathways that control endothelial cell (EC) permeability, leukocyte adhesion and inflammation are pivotal for atherosclerosis initiation and progression. Here we demonstrate that the Sterile-20-like mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), which has been implicated in inflammation, is abundantly expressed in ECs and in atherosclerotic plaques from mice and humans. On the basis of endothelial-specific MAP4K4 gene silencing and gene ablation experiments in Apoe(-/-) mice, we show that MAP4K4 in ECs markedly promotes Western diet-induced aortic macrophage accumulation and atherosclerotic plaque development. Treatment of Apoe(-/-) and Ldlr(-/-) mice with a selective small-molecule MAP4K4 inhibitor also markedly reduces atherosclerotic lesion area. MAP4K4 silencing in cultured ECs attenuates cell surface adhesion molecule expression while reducing nuclear localization and activity of NFκB, which is critical for promoting EC activation and atherosclerosis. Taken together, these results reveal that MAP4K4 is a key signalling node that promotes immune cell recruitment in atherosclerosis.
Asunto(s)
Aterosclerosis/metabolismo , Células Endoteliales/metabolismo , Inflamación/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Enfermedades Vasculares/metabolismo , Aminopiridinas/farmacología , Animales , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerosis/genética , Regulación de la Expresión Génica/fisiología , Inflamación/genética , Macrófagos , Masculino , Ratones , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Receptores de LDL/genética , Receptores de LDL/metabolismo , Enfermedades Vasculares/genética , Quinasa de Factor Nuclear kappa BRESUMEN
Sirtuin 3 (SIRT3), an important regulator of energy metabolism and lipid oxidation, is induced in fasted liver mitochondria and implicated in metabolic syndrome. In fasted liver, SIRT3-mediated increases in substrate flux depend on oxidative phosphorylation (OXPHOS), but precisely how OXPHOS meets the challenge of increased substrate oxidation in fasted liver remains unclear. Here, we show that liver mitochondria in fasting mice adapt to the demand of increased substrate oxidation by increasing their OXPHOS efficiency. In response to cAMP signaling, SIRT3 deacetylated and activated leucine-rich protein 130 (LRP130; official symbol, LRPPRC), promoting a mitochondrial transcriptional program that enhanced hepatic OXPHOS. Using mass spectrometry, we identified SIRT3-regulated lysine residues in LRP130 that generated a lysine-to-arginine (KR) mutant of LRP130 that mimics deacetylated protein. Compared with wild-type LRP130 protein, expression of the KR mutant increased mitochondrial transcription and OXPHOS in vitro. Indeed, even when SIRT3 activity was abolished, activation of mitochondrial transcription and OXPHOS by the KR mutant remained robust, further highlighting the contribution of LRP130 deacetylation to increased OXPHOS in fasted liver. These data establish a link between nutrient sensing and mitochondrial transcription that regulates OXPHOS in fasted liver and may explain how fasted liver adapts to increased substrate oxidation.
Asunto(s)
Mitocondrias Hepáticas/metabolismo , Fosforilación Oxidativa , Sirtuina 3/metabolismo , Alelos , Secuencia de Aminoácidos , Animales , Arginina/metabolismo , Citrato (si)-Sintasa/metabolismo , AMP Cíclico/metabolismo , Metabolismo Energético/genética , Alimentos , Glucagón/metabolismo , Hepatocitos/citología , Hígado/metabolismo , Lisina/metabolismo , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Transcripción GenéticaRESUMEN
Obesity places major demands on the protein folding capacity of the endoplasmic reticulum (ER), resulting in ER stress, a condition that promotes hepatic insulin resistance and steatosis. Here we identify the transcription factor, Kruppel-like factor 15 (KLF15), as an essential mediator of ER stress-induced insulin resistance in the liver. Mice with a targeted deletion of KLF15 exhibit increased hepatic ER stress, inflammation, and JNK activation compared to WT mice; however, KLF15 (-/-) mice are protected against hepatic insulin resistance and fatty liver under high-fat feeding conditions and in response to pharmacological induction of ER stress. The mammalian target of rapamycin complex 1 (mTORC1), a key regulator of cellular energy homeostasis, has been shown to cooperate with ER stress signaling pathways to promote hepatic insulin resistance and lipid accumulation. We find that the uncoupling of ER stress and insulin resistance in KLF15 (-/-) liver is associated with the maintenance of a low energy state characterized by decreased mTORC1 activity, increased AMPK phosphorylation and PGC-1α expression and activation of autophagy, an intracellular degradation process that enhances hepatic insulin sensitivity. Furthermore, in primary hepatocytes, KLF15 deficiency markedly inhibits activation of mTORC1 by amino acids and insulin, suggesting a mechanism by which KLF15 controls mTORC1-mediated insulin resistance. This study establishes KLF15 as an important molecular link between ER stress and insulin action.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Estrés del Retículo Endoplásmico , Resistencia a la Insulina , Hígado/metabolismo , Factores de Transcripción/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Proteínas de Unión al ADN/genética , Grasas de la Dieta/efectos adversos , Grasas de la Dieta/farmacología , Hígado Graso/inducido químicamente , Hígado Graso/genética , Hígado Graso/metabolismo , Hígado Graso/patología , Humanos , Factores de Transcripción de Tipo Kruppel , Hígado/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Noqueados , Complejos Multiproteicos , Obesidad/inducido químicamente , Obesidad/genética , Obesidad/metabolismo , Obesidad/patología , Fosforilación , Serina-Treonina Quinasas TOR , Factores de Transcripción/genéticaRESUMEN
Cockayne syndrome (CS) is a devastating autosomal recessive disease characterized by neurodegeneration, cachexia, and accelerated aging. 80% of the cases are caused by mutations in the CS complementation group B (CSB) gene known to be involved in DNA repair and transcription. Recent evidence indicates that CSB is present in mitochondria, where it associates with mitochondrial DNA (mtDNA). We report an increase in metabolism in the CSB(m/m) mouse model and CSB-deficient cells. Mitochondrial content is increased in CSB-deficient cells, whereas autophagy is down-regulated, presumably as a result of defects in the recruitment of P62 and mitochondrial ubiquitination. CSB-deficient cells show increased free radical production and an accumulation of damaged mitochondria. Accordingly, treatment with the autophagic stimulators lithium chloride or rapamycin reverses the bioenergetic phenotype of CSB-deficient cells. Our data imply that CSB acts as an mtDNA damage sensor, inducing mitochondrial autophagy in response to stress, and that pharmacological modulators of autophagy are potential treatment options for this accelerated aging phenotype.
Asunto(s)
Autofagia , ADN Helicasas/fisiología , Enzimas Reparadoras del ADN/fisiología , Mitocondrias/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Transporte de Membrana Mitocondrial , Poro de Transición de la Permeabilidad Mitocondrial , Proteínas de Unión a Poli-ADP-Ribosa , Especies Reactivas de Oxígeno/metabolismoRESUMEN
OBJECTIVE: The peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1 family of transcriptional coactivators controls hepatic function by modulating the expression of key metabolic enzymes. Hepatic gain of function and complete genetic ablation of PGC-1alpha show that this coactivator is important for activating the programs of gluconeogenesis, fatty acid oxidation, oxidative phosphorylation, and lipid secretion during times of nutrient deprivation. However, how moderate changes in PGC-1alpha activity affect metabolism and energy homeostasis has yet to be determined. RESEARCH DESIGN AND METHODS: To identify key metabolic pathways that may be physiologically relevant in the context of reduced hepatic PGC-1alpha levels, we used the Cre/Lox system to create mice heterozygous for PGC-1alpha specifically within the liver (LH mice). RESULTS: These mice showed fasting hepatic steatosis and diminished ketogenesis associated with decreased expression of genes involved in mitochondrial beta-oxidation. LH mice also exhibited high circulating levels of triglyceride that correlated with increased expression of genes involved in triglyceride-rich lipoprotein assembly. Concomitant with defects in lipid metabolism, hepatic insulin resistance was observed both in LH mice fed a high-fat diet as well as in primary hepatocytes. CONCLUSIONS: These data highlight both the dose-dependent and long-term effects of reducing hepatic PGC-1alpha levels, underlining the importance of tightly regulated PGC-1alpha expression in the maintenance of lipid homeostasis and glucose metabolism.