RESUMEN
MAF1 is a global repressor of RNA polymerase III transcription that regulates the expression of highly abundant noncoding RNAs in response to nutrient availability and cellular stress. Thus, MAF1 function is thought to be important for metabolic economy. Here we show that a whole-body knockout of Maf1 in mice confers resistance to diet-induced obesity and nonalcoholic fatty liver disease by reducing food intake and increasing metabolic inefficiency. Energy expenditure in Maf1(-/-) mice is increased by several mechanisms. Precursor tRNA synthesis was increased in multiple tissues without significant effects on mature tRNA levels, implying increased turnover in a futile tRNA cycle. Elevated futile cycling of hepatic lipids was also observed. Metabolite profiling of the liver and skeletal muscle revealed elevated levels of many amino acids and spermidine, which links the induction of autophagy in Maf1(-/-) mice with their extended life span. The increase in spermidine was accompanied by reduced levels of nicotinamide N-methyltransferase, which promotes polyamine synthesis, enables nicotinamide salvage to regenerate NAD(+), and is associated with obesity resistance. Consistent with this, NAD(+) levels were increased in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences.
Asunto(s)
Proteínas Represoras/genética , Animales , Autofagia/genética , Ingestión de Alimentos/genética , Metabolismo Energético/genética , Metabolismo de los Lípidos/genética , Longevidad/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/genética , Obesidad/genética , ARN de Transferencia/metabolismo , Espermidina/metabolismoRESUMEN
BACKGROUND: Fetal adaptations to high fat (HF) diet in utero (IU) that may predispose to Metabolic Syndrome (MetS) in adulthood include changes in fetal hepatic gene expression. Studies were performed to determine whether maternal exposure to HF diet at different stages during pregnancy had different effects on the fetus, including hepatic gene expression. METHODS: Female wild type mice were fed either a HF or breeding chow (C) for 2 wks prior to mating. The experimental groups were composed of embryonic day (e) 18.5 fetuses obtained from WT female mice that were fed HF (HF, 35.5% fat) or breeding chow (C, 9.5% fat) for 2 wk before mating until e9.5 of pregnancy (periconception-midpregnancy). At e9.5 dams were switched to the opposite diet (C-HF or HF-C). RESULTS: Exposure to HF diet throughout pregnancy reduced maternal weight gain compared to C diet (p < 0.02 HF vs. C). HF-C dams had significantly decreased adiponectin levels and litter size when compared to C-HF (p < 0.02 HF-C vs C-HF). Independent of the timing of exposure to HF, fetal weight and length were significantly decreased when compared to C diet (HF, C-HF and HF-C vs. C p < 0.02). HF diet during the second half of pregnancy increased expression of genes in the fetal liver associated with fetal growth (C-HF vs C p < 0.001), glucose production (C-HF vs C p < 0.04), oxidative stress and inflammation (C-HF vs C p < 0.01) compared to C diet. CONCLUSIONS: This model defines that there are critical periods during gestation in which the fetus is actively shaped by the environment. Early exposure to a HF diet determines litter size while exposure to HF during the second half of pregnancy leads to dysregulation of expression of key genes responsible for fetal growth, hepatic glucose production and oxidative stress. These findings underscore the importance of future studies designed to clarify how these critical periods may influence future risk of developing MetS later in life.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Desarrollo Fetal , Retardo del Crecimiento Fetal/etiología , Hiperglucemia/etiología , Fenómenos Fisiologicos Nutricionales Maternos , Síndrome Metabólico/etiología , Estrés Oxidativo , Adiponectina/sangre , Animales , Animales no Consanguíneos , Cruzamientos Genéticos , Femenino , Retardo del Crecimiento Fetal/inmunología , Retardo del Crecimiento Fetal/metabolismo , Peso Fetal , Regulación del Desarrollo de la Expresión Génica , Gluconeogénesis , Transportador de Glucosa de Tipo 4/genética , Hiperglucemia/embriología , Hiperglucemia/inmunología , Hiperglucemia/metabolismo , Tamaño de la Camada , Hígado/embriología , Hígado/inmunología , Hígado/metabolismo , Síndrome Metabólico/embriología , Síndrome Metabólico/inmunología , Síndrome Metabólico/metabolismo , Ratones MutantesRESUMEN
OBJECTIVE: Data from animal models show that in utero exposure to a maternal high-fat diet (HFD) renders susceptibility of these offspring to the adult onset of metabolic syndrome. We and others have previously shown that epigenetic modifications to histones may serve as a molecular memory of the in utero exposure, rendering the risk of adult disease. Because mice heterozygous for the Glut4 gene (insulin sensitive glucose transporter) born to wild-type (WT) mothers demonstrate exacterbated metabolic syndrome when exposed to an HFD in utero, we sought to analyze the genome-wide epigenetic changes that occur in the fetal liver in susceptible offspring. STUDY DESIGN: WT and Glut4(+/-) (G4(+/-)) offspring of WT mothers that were exposed either to a control or an HFD in utero were studied. Immunoblotting was used to measure hepatic histone modifications of fetal and 5-week animals. Chromatin immunoprecipitation (ChIP) followed by hybridization to chip arrays (ChIP-on-chip) was used to detect genome-wide changes of histone modifications with HFD exposure. RESULTS: We found that levels of hepatic H3K14ac and H3K9me3 significantly increased with HFD exposure in WT and G4(+/-) fetal and 5-week offspring. Pathway analysis of our ChIP-on-chip data revealed differential H3K14ac and H3K9me3 enrichment along pathways that regulate lipid metabolism, specifically in the promoter regions of Pparg, Ppara, Rxra, and Rora. CONCLUSION: We conclude that HFD exposure in utero is associated with functional alterations to fetal hepatic histone modifications in both WT and G4(+/-) offspring, some of which persist up to 5 weeks of age.
Asunto(s)
Grasas de la Dieta/farmacología , Código de Histonas/efectos de los fármacos , Histonas/genética , Hígado/embriología , Efectos Tardíos de la Exposición Prenatal/genética , Fenómenos Fisiologicos de la Nutrición Prenatal/genética , Acetilación/efectos de los fármacos , Animales , Hibridación Genómica Comparativa , Dieta Alta en Grasa/efectos adversos , Epigénesis Genética/efectos de los fármacos , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Predisposición Genética a la Enfermedad , Transportador de Glucosa de Tipo 4/genética , Código de Histonas/genética , Hígado/enzimología , Síndrome Metabólico/genética , Ratones , Modelos Animales , EmbarazoRESUMEN
In this study, a tandem LC-MS (Waters Xevo TQ) MRM-based MS method was developed for rapid, broad profiling of hydrophilic metabolites from biological samples, in either positive or negative ion modes without the need for an ion pairing reagent, using a reversed-phase pentafluorophenylpropyl (PFPP) column. The developed method was successfully applied to analyze various biological samples from C57BL/6 mice, including urine, duodenum, liver, plasma, kidney, heart, and skeletal muscle. As result, a total 112 of hydrophilic metabolites were detected within 8 min of running time to obtain a metabolite profile of the biological samples. The analysis of this number of hydrophilic metabolites is significantly faster than previous studies. Classification separation for metabolites from different tissues was globally analyzed by PCA, PLS-DA and HCA biostatistical methods. Overall, most of the hydrophilic metabolites were found to have a "fingerprint" characteristic of tissue dependency. In general, a higher level of most metabolites was found in urine, duodenum, and kidney. Altogether, these results suggest that this method has potential application for targeted metabolomic analyzes of hydrophilic metabolites in a wide ranges of biological samples.
Asunto(s)
Cromatografía Liquida/instrumentación , Cromatografía Liquida/métodos , Redes y Vías Metabólicas , Metabolómica/métodos , Espectrometría de Masas en Tándem/instrumentación , Espectrometría de Masas en Tándem/métodos , Animales , Ratones , Ratones Endogámicos C57BL , Análisis Multivariante , Silanos/químicaRESUMEN
The elucidation of extra-nuclear lysine acetylation has been of growing interest, as the cosubstrate for acetylation, acetyl CoA, is at a key metabolic intersection. Our hypothesis was that mitochondrial and cytoplasmic protein acetylation may be part of a fasted/re-fed feedback control system for the regulation of the metabolic network in fuel switching, where acetyl CoA would be provided by fatty acid oxidation, or glycolysis, respectively. To test this, we characterized the mitochondrial and cytoplasmic acetylome in various organs that have a high metabolic rate relative to their mass, and/or switch fuels, under fasted and re-fed conditions (brain, kidney, liver, skeletal muscle, heart muscle, white and brown adipose tissues). Using immunoprecipitation, coupled with LC-MS/MS label free quantification, we show there is a dramatic variation in global quantitative profiles of acetylated proteins from different organs. In total, 733 acetylated peptides from 337 proteins were identified and quantified, out of which 31 acetylated peptides from the metabolic proteins that may play organ-specific roles were analyzed in detail. Results suggest that fasted/re-fed acetylation changes coordinated by organ-specific (de)acetylases in insulin-sensitive versus -insensitive organs may underlie fuel use and switching. Characterization of the tissue-specific acetylome should increase understanding of metabolic conditions wherein normal fuel switching is disrupted, such as in Type II diabetes.
Asunto(s)
Acetilcoenzima A/metabolismo , Proteínas/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Acetilación , Secuencia de Aminoácidos , Animales , Cromatografía Liquida , Citoplasma/metabolismo , Metabolismo Energético , Ayuno/metabolismo , Inmunoprecipitación , Lisina/metabolismo , Masculino , Redes y Vías Metabólicas , Ratones , Ratones Endogámicos , Proteínas Mitocondriales/análisis , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares , Datos de Secuencia Molecular , Especificidad de Órganos , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/metabolismo , Proteoma/análisis , Espectrometría de Masas en TándemRESUMEN
Studies were conducted to determine whether maternal substrate utilization during pregnancy affects fetal growth and predisposes offspring to metabolic disease. Female wild-type (WT) and glucose transporter 4 heterozygous mice (G4+/-, a model of altered peripheral substrate utilization) were fed high-fat diet (HFD, 35.5% fat) or control chow (C, 9.5% fat) for 2 wk before mating, throughout pregnancy and lactation (IU/L). WT HFD females exhibited increased serum nonesterified fatty acid and lactate levels and increased hepatic mRNA expression of peroxisome proliferator-activated receptor gamma coactivator-1-beta and SREBP-1c, consistent with increased lipogenesis. G4+/- HFD females exhibited enhanced lipid clearance, and exposure to HFD did not increase hepatic gene expression. HFD independent of maternal genotype decreased fetal growth and birth weight. WT offspring were weaned onto a low-fat diet (5.6% fat). Male offspring of WT mothers exposed to HFD exhibited "catch-up" growth accompanied by increased adiposity, impaired glucose tolerance, and insulin sensitivity. In contrast, male offspring of G4+/- HFD mothers did not exhibit any characteristics of metabolic syndrome. These data suggest that differences in maternal substrate utilization influence offspring metabolic phenotype.
Asunto(s)
Grasas de la Dieta/metabolismo , Fenómenos Fisiologicos Nutricionales Maternos , Síndrome Metabólico/metabolismo , Fenómenos Fisiologicos de la Nutrición Prenatal , Animales , Peso Corporal , Ingestión de Alimentos , Femenino , Desarrollo Fetal/fisiología , Genotipo , Glucosa/metabolismo , Insulina/metabolismo , Resistencia a la Insulina/fisiología , Metabolismo de los Lípidos , Masculino , Ratones , EmbarazoRESUMEN
PURPOSE: Assessing whole-body radiation injury and absorbed dose is essential for remediation efforts following accidental or deliberate exposure in medical, industrial, military, or terrorist incidents. We hypothesize that variations in specific metabolite concentrations extracted from blood plasma would correlate with whole-body radiation injury and dose. METHODS AND MATERIALS: Groups of C57BL/6 mice (n=12 per group) were exposed to 0, 2, 4, 8, and 10.4 Gy of whole-body gamma radiation. At 24 hours after treatment, all animals were euthanized, and both plasma and liver biopsy samples were obtained, the latter being used to identify a distinct hepatic radiation injury response within plasma. A semiquantitative, untargeted metabolite/lipid profile was developed using gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry, which identified 354 biochemical compounds. A second set of C57BL/6 mice (n=6 per group) were used to assess a subset of identified plasma markers beyond 24 hours. RESULTS: We identified a cohort of 37 biochemical compounds in plasma that yielded the optimal separation of the irradiated sample groups, with the most correlated metabolites associated with pyrimidine (positively correlated) and tryptophan (negatively correlated) metabolism. The latter were predominantly associated with indole compounds, and there was evidence that these were also correlated between liver and plasma. No evidence of saturation as a function of dose was observed, as has been noted for studies involving metabolite analysis of urine. CONCLUSIONS: Plasma profiling of specific metabolites related to pyrimidine and tryptophan pathways can be used to differentiate whole-body radiation injury and dose response. As the tryptophan-associated indole compounds have their origin in the intestinal microbiome and subsequently the liver, these metabolites particularly represent an attractive marker for radiation injury within blood plasma.
Asunto(s)
Proteínas Bacterianas/sangre , Intestinos/microbiología , Lípidos/sangre , Proteoma/análisis , Traumatismos por Radiación/sangre , Traumatismos por Radiación/microbiología , Irradiación Corporal Total/efectos adversos , Animales , Biomarcadores/sangre , Relación Dosis-Respuesta en la Radiación , Intestinos/efectos de la radiación , Masculino , Metaboloma , Ratones , Ratones Endogámicos C57BL , Dosis de Radiación , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Intrauterine (IU) malnutrition could alter pancreatic development. In this study, we describe the effects of high-fat diet (HFD) during pregnancy on fetal growth and pancreatic morphology in an 'at risk' animal model of metabolic disease, the glucose transporter 4 (GLUT4) heterozygous mouse (G4+/-). WT female mice mated with G4+/- males were fed HFD or control diet (CD) for 2 weeks before mating and throughout pregnancy. At embryonic day 18.5, fetuses were killed and pancreata isolated for analysis of morphology and expression of genes involved in insulin (INS) cell development, proliferation, apoptosis, glucose transport and function. Compared with WT CD, WT HFD fetal pancreata had a 2.4-fold increase in the number of glucagon (GLU) cells (P=0.023). HFD also increased GLU cell size by 18% in WT pancreata compared with WT CD. Compared with WT CD, G4+/- CD had an increased number of INS cells and decreased INS and GLU cell size. Compared with G4+/- CD, G4+/- HFD fetuses had increased pancreatic gene expression of Igf2, a mitogen and inhibitor of apoptosis. The expression of genes involved in proliferation, apoptosis, glucose transport, and INS secretion was not altered in WT HFD compared with G4+/- HFD pancreata. In contrast to WT HFD pancreata, HFD exposure did not alter pancreatic islet morphology in fetuses with GLUT4 haploinsufficiency; this may be mediated in part by increased Igf2 expression. Thus, interactions between IU diet and fetal genetics may play a critical role in the developmental origins of health and disease.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Transportador de Glucosa de Tipo 4/genética , Páncreas/embriología , Animales , Femenino , Desarrollo Fetal , Glucagón/metabolismo , Factor II del Crecimiento Similar a la Insulina/biosíntesis , Células Secretoras de Insulina/fisiología , Masculino , Ratones , Páncreas/metabolismo , Embarazo , Efectos Tardíos de la Exposición PrenatalRESUMEN
Insulin integrates hepatic glucose and lipid metabolism, directing nutrients to storage as glycogen and triglyceride. In type 2 diabetes, levels of the former are low and the latter are exaggerated, posing a pathophysiologic and therapeutic conundrum. A branching model of insulin signalling, with FoxO1 presiding over glucose production and Srebp-1c regulating lipogenesis, provides a potential explanation. Here we illustrate an alternative mechanism that integrates glucose production and lipogenesis under the unifying control of FoxO. Liver-specific ablation of three FoxOs (L-FoxO1,3,4) prevents the induction of glucose-6-phosphatase and the repression of glucokinase during fasting, thus increasing lipogenesis at the expense of glucose production. We document a similar pattern in the early phases of diet-induced insulin resistance, and propose that FoxOs are required to enable the liver to direct nutritionally derived carbons to glucose versus lipid metabolism. Our data underscore the heterogeneity of hepatic insulin resistance during progression from the metabolic syndrome to overt diabetes, and the conceptual challenge of designing therapies that curtail glucose production without promoting hepatic lipid accumulation.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Factores de Transcripción Forkhead/metabolismo , Glucosa/metabolismo , Lipogénesis , Hígado/metabolismo , Animales , Proteínas de Ciclo Celular , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/genética , Ayuno/metabolismo , Proteína Forkhead Box O1 , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Glucoquinasa/genética , Glucoquinasa/metabolismo , Glucosa-6-Fosfatasa/genética , Glucosa-6-Fosfatasa/metabolismo , Insulina/metabolismo , Metabolismo de los Lípidos , Hígado/enzimología , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Genetic and environmental factors, including the in utero environment, contribute to Metabolic Syndrome. Exposure to high fat diet exposure in utero and lactation increases incidence of Metabolic Syndrome in offspring. Using GLUT4 heterozygous (G4+/-) mice, genetically predisposed to Type 2 Diabetes Mellitus, and wild-type littermates we demonstrate genotype specific differences to high fat in utero and lactation. High fat in utero and lactation increased adiposity and impaired insulin and glucose tolerance in both genotypes. High fat wild type offspring had increased serum glucose and PAI-1 levels and decreased adiponectin at 6 wks of age compared to control wild type. High fat G4+/- offspring had increased systolic blood pressure at 13 wks of age compared to all other groups. Potential fetal origins of adult Metabolic Syndrome were investigated. Regardless of genotype, high fat in utero decreased fetal weight and crown rump length at embryonic day 18.5 compared to control. Hepatic expression of genes involved in glycolysis, gluconeogenesis, oxidative stress and inflammation were increased with high fat in utero. Fetal serum glucose levels were decreased in high fat G4+/- compared to high fat wild type fetuses. High fat G4+/-, but not high fat wild type fetuses, had increased levels of serum cytokines (IFN-γ, MCP-1, RANTES and M-CSF) compared to control. This data demonstrates that high fat during pregnancy and lactation increases Metabolic Syndrome male offspring and that heterozygous deletion of GLUT4 augments susceptibility to increased systolic blood pressure. Fetal adaptations to high fat in utero that may predispose to Metabolic Syndrome in adulthood include changes in fetal hepatic gene expression and alterations in circulating cytokines. These results suggest that the interaction between in utero-perinatal environment and genotype plays a critical role in the developmental origin of health and disease.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Regulación del Desarrollo de la Expresión Génica/fisiología , Síndrome Metabólico/etiología , Efectos Tardíos de la Exposición Prenatal/patología , Adiponectina/metabolismo , Adiposidad/genética , Análisis de Varianza , Animales , Glucemia/metabolismo , Presión Sanguínea/fisiología , Composición Corporal/fisiología , Cruzamientos Genéticos , Citocinas/sangre , Femenino , Peso Fetal , Regulación del Desarrollo de la Expresión Génica/genética , Genotipo , Transportador de Glucosa de Tipo 4/genética , Heterocigoto , Resistencia a la Insulina/genética , Hígado/metabolismo , Masculino , Ratones , Embarazo , Reacción en Cadena en Tiempo Real de la Polimerasa , Serpina E2/metabolismoRESUMEN
BACKGROUND: FAAH (fatty acid amide hydrolase), primarily expressed in the liver, hydrolyzes the endocannabinoids fatty acid ethanolamides (FAA). Human FAAH gene mutations are associated with increased body weight and obesity. In our present study, using targeted metabolite and lipid profiling, and new global acetylome profiling methodologies, we examined the role of the liver on fuel and energy homeostasis in whole body FAAH(-/-) mice. METHODOLOGY/PRINCIPAL FINDINGS: FAAH(-/-) mice exhibit altered energy homeostasis demonstrated by decreased oxygen consumption (Indirect calorimetry). FAAH(-/-) mice are hyperinsulinemic and have adipose, skeletal and hepatic insulin resistance as indicated by stable isotope phenotyping (SIPHEN). Fed state skeletal muscle and liver triglyceride levels was increased 2-3 fold, while glycogen was decreased 42% and 57% respectively. Hepatic cholesterol synthesis was decreased 22% in FAAH(-/-) mice. Dysregulated hepatic FAAH(-/-) lysine acetylation was consistent with their metabolite profiling. Fasted to fed increases in hepatic FAAH(-/-) acetyl-CoA (85%, p<0.01) corresponded to similar increases in citrate levels (45%). Altered FAAH(-/-) mitochondrial malate dehydrogenase (MDH2) acetylation, which can affect the malate aspartate shuttle, was consistent with our observation of a 25% decrease in fed malate and aspartate levels. Decreased fasted but not fed dihydroxyacetone-P and glycerol-3-P levels in FAAH(-/-) mice was consistent with a compensating contribution from decreased acetylation of fed FAAH(-/-) aldolase B. Fed FAAH(-/-) alcohol dehydrogenase (ADH) acetylation was also decreased. CONCLUSIONS/SIGNIFICANCE: Whole body FAAH deletion contributes to a pre-diabetic phenotype by mechanisms resulting in impairment of hepatic glucose and lipid metabolism. FAAH(-/-) mice had altered hepatic lysine acetylation, the pattern sharing similarities with acetylation changes reported with chronic alcohol treatment. Dysregulated hepatic lysine acetylation seen with impaired FAA hydrolysis could support the liver's role in fostering the pre-diabetic state, and may reflect part of the mechanism underlying the hepatic effects of endocannabinoids in alcoholic liver disease mouse models.
Asunto(s)
Amidohidrolasas/metabolismo , Metabolismo Energético , Homeostasis , Metabolismo de los Lípidos , Lisina/metabolismo , Obesidad/enzimología , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Acetilación , Amidohidrolasas/genética , Animales , Regulación Enzimológica de la Expresión Génica , Glucosa/genética , Glucosa/metabolismo , Humanos , Hígado , Lisina/genética , Ratones , Ratones Noqueados , Obesidad/genéticaRESUMEN
Acute activation of the serine-threonine kinase Akt is cardioprotective and increases glucose uptake, at least in part, through enhanced expression of GLUT4 on the sarcolemma. The effects of chronic Akt activation on glucose uptake in the heart remain unclear. To address this issue, we examined the effects of chronic Akt activation on glucose uptake, glycogen storage, and relevant glucose transporters in the hearts of transgenic mice. We found that chronic cardiac activation of Akt led to a substantial increase in the rate of basal glucose uptake (P < 0.05) but blunted the response to insulin (1.9 vs. 18.1-fold increase compared with baseline) using NMR in ex vivo perfused heart. Basal glucose uptake was also increased in Akt transgenic mice in vivo (P < 0.005). These changes were associated with an increase on glycogen deposition, examined with histochemical staining, biochemical (>6-fold, P < 0.001) and in vivo radioactive (5-fold, P < 0.01) assays. Studies in chimeric hearts of female X-linked transgenic Akt mice suggested that increased glycogen deposition occurred as a cell autonomous effect of transgene expression. Interestingly, although sarcolemmal GLUT1 was not significantly altered, chronic Akt activation actually decreased plasma membrane GLUT4. Moreover, intracellular pools of GLUT1 were modestly reduced, whereas intracellular GLUT4 was substantially reduced. It seems likely that neither GLUT1 nor GLUT4 explains the increase in basal glucose uptake but that these reductions contribute to the loss of insulin responsiveness that we observed. These data demonstrate that chronic Akt activation increases basal glucose uptake and glycogen deposition while inhibiting the response to insulin.