RESUMO
Quantitative subcellular metabolomic measurements can explain the roles of metabolites in cellular processes but are subject to multiple confounding factors. We developed stable isotope labeling of essential nutrients in cell culture-subcellular fractionation (SILEC-SF), which uses isotope-labeled internal standard controls that are present throughout fractionation and processing to quantify acyl-coenzyme A (acyl-CoA) thioesters in subcellular compartments by liquid chromatography-mass spectrometry. We tested SILEC-SF in a range of sample types and examined the compartmentalized responses to oxygen tension, cellular differentiation, and nutrient availability. Application of SILEC-SF to the challenging analysis of the nuclear compartment revealed a nuclear acyl-CoA profile distinct from that of the cytosol, with notable nuclear enrichment of propionyl-CoA. Using isotope tracing, we identified the branched chain amino acid isoleucine as a major metabolic source of nuclear propionyl-CoA and histone propionylation, thus revealing a new mechanism of crosstalk between metabolism and the epigenome.
Assuntos
Acil Coenzima A/metabolismo , Compartimento Celular , Núcleo Celular/metabolismo , Metabolismo Energético , Histonas/metabolismo , Metabolômica , Processamento de Proteína Pós-Traducional , Animais , Diferenciação Celular , Cromatografia Líquida , Citosol/metabolismo , Epigênese Genética , Células Hep G2 , Humanos , Isoleucina , Metaboloma , Camundongos , Mitocôndrias/metabolismo , Oxigênio/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Insulin increases glucose uptake into adipose tissue and muscle by increasing trafficking of the glucose transporter Glut4. In cultured adipocytes, the exocytosis of Glut4 relies on activation of the small G protein RalA by insulin, via inhibition of its GTPase activating complex RalGAP. Here, we evaluate the role of RalA in glucose uptake in vivo with specific chemical inhibitors and by generation of mice with adipocyte-specific knockout of RalGAPB. RalA was profoundly activated in brown adipose tissue after feeding, and its inhibition prevented Glut4 exocytosis. RalGAPB knockout mice with diet-induced obesity were protected from the development of metabolic disease due to increased glucose uptake into brown fat. Thus, RalA plays a crucial role in glucose transport in adipose tissue in vivo.
Assuntos
Tecido Adiposo Marrom/metabolismo , Glucose/metabolismo , Homeostase , Proteínas ral de Ligação ao GTP/metabolismo , Células 3T3-L1 , Tecido Adiposo Marrom/patologia , Animais , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Deleção de Genes , Glucose/genética , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Camundongos , Camundongos Knockout , Proteínas ral de Ligação ao GTP/genéticaRESUMO
N-acetylaspartate (NAA) is synthesized by aspartate N-acetyltransferase (gene: Nat8l) from acetyl-coenzyme A and aspartate. In the brain, NAA is considered an important energy metabolite for lipid synthesis. However, the role of NAA in peripheral tissues remained elusive. Therefore, we characterized the metabolic phenotype of knockout (ko) and adipose tissue-specific (ako) Nat8l-ko mice as well as NAA-supplemented mice on various diets. We identified an important role of NAA availability in the brain during adolescence, as 75% of Nat8l-ko mice died on fat-free diet (FFD) after weaning but could be rescued by NAA supplementation. In adult life, NAA deficiency promotes a beneficial metabolic phenotype, as Nat8l-ko and Nat8l-ako mice showed reduced body weight, increased energy expenditure, and improved glucose tolerance on chow, high-fat, and FFDs. Furthermore, Nat8l-deficient adipocytes exhibited increased mitochondrial respiration, ATP synthesis, and an induction of browning. Conversely, NAA-treated wild-type mice showed reduced adipocyte respiration and lipolysis and increased de novo lipogenesis, culminating in reduced energy expenditure, glucose tolerance, and insulin sensitivity. Mechanistically, our data point to a possible role of NAA as modulator of pancreatic insulin secretion and suggest NAA as a critical energy metabolite for adipocyte and whole-body energy homeostasis.-Hofer, D. C., Zirkovits, G., Pelzmann, H. J., Huber, K., Pessentheiner, A. R., Xia, W., Uno, K., Miyazaki, T., Kon, K., Tsuneki, H., Pendl, T., Al Zoughbi, W., Madreiter-Sokolowski, C. T., Trausinger, G., Abdellatif, M., Schoiswohl, G., Schreiber, R., Eisenberg, T., Magnes, C., Sedej, S., Eckhardt, M., Sasahara, M., Sasaoka, T., Nitta, A., Hoefler, G., Graier, W. F., Kratky, D., Auwerx, J., Bogner-Strauss, J. G. N-acetylaspartate availability is essential for juvenile survival on fat-free diet and determines metabolic health.
Assuntos
Ácido Aspártico/análogos & derivados , Acetilcoenzima A/metabolismo , Acetiltransferases/metabolismo , Adipócitos/metabolismo , Animais , Ácido Aspártico/metabolismo , Encéfalo/metabolismo , Dieta com Restrição de Gorduras , Metabolismo Energético/fisiologia , Resistência à Insulina/fisiologia , Lipólise/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismoRESUMO
Lysosomal acid lipase (LAL) is the only known enzyme, which hydrolyzes cholesteryl esters and triacylglycerols in lysosomes of multiple cells and tissues. Here, we explored the role of LAL in brown adipose tissue (BAT). LAL-deficient (Lal-/-) mice exhibit markedly reduced UCP1 expression in BAT, modified BAT morphology with accumulation of lysosomes, and mitochondrial dysfunction, consequently leading to regular hypothermic events in mice kept at room temperature. Cold exposure resulted in reduced lipid uptake into BAT, thereby aggravating dyslipidemia and causing life threatening hypothermia in Lal-/- mice. Linking LAL as a potential regulator of lipoprotein lipase activity, we found Angptl4 mRNA expression upregulated in BAT. Our data demonstrate that LAL is critical for shuttling fatty acids derived from circulating lipoproteins to BAT during cold exposure. We conclude that inhibited lysosomal lipid hydrolysis in BAT leads to impaired thermogenesis in Lal-/- mice.
Assuntos
Tecido Adiposo Marrom/metabolismo , Ácidos Graxos/metabolismo , Esterol Esterase/metabolismo , Termogênese , Acetilcoenzima A/metabolismo , Adipócitos Marrons/metabolismo , Tecido Adiposo Marrom/ultraestrutura , Animais , Autofagia , Temperatura Corporal , Carnitina/análogos & derivados , Carnitina/metabolismo , Temperatura Baixa , Progressão da Doença , Dislipidemias/metabolismo , Dislipidemias/patologia , Metabolismo Energético , Glucose/metabolismo , Hipotermia Induzida , Gotículas Lipídicas/metabolismo , Lipólise , Masculino , Camundongos Endogâmicos C57BL , Músculos/metabolismo , Oxirredução , Consumo de Oxigênio , Esterol Esterase/deficiência , Proteína Desacopladora 1/metabolismoRESUMO
The ability to adapt cellular metabolism to nutrient availability is critical for survival. The liver plays a central role in the adaptation to starvation by switching from glucose-consuming processes and lipid synthesis to providing energy substrates like glucose to the organism. Here we report a previously unrecognized role of the tumor suppressor p53 in the physiologic adaptation to food withdrawal. We found that starvation robustly increases p53 protein in mouse liver. This induction was posttranscriptional and mediated by a hepatocyte-autonomous and AMP-activated protein kinase-dependent mechanism. p53 stabilization was required for the adaptive expression of genes involved in amino acid catabolism. Indeed, acute deletion of p53 in livers of adult mice impaired hepatic glycogen storage and induced steatosis. Upon food withdrawal, p53-deleted mice became hypoglycemic and showed defects in the starvation-associated utilization of hepatic amino acids. In summary, we provide novel evidence for a p53-dependent integration of acute changes of cellular energy status and the metabolic adaptation to starvation. Because of its tumor suppressor function, p53 stabilization by starvation could have implications for both metabolic and oncological diseases of the liver.-Prokesch, A., Graef, F. A., Madl, T., Kahlhofer, J., Heidenreich, S., Schumann, A., Moyschewitz, E., Pristoynik, P., Blaschitz, A., Knauer, M., Muenzner, M., Bogner-Strauss, J. G., Dohr, G., Schulz, T. J., Schupp, M. Liver p53 is stabilized upon starvation and required for amino acid catabolism and gluconeogenesis.
Assuntos
Privação de Alimentos/fisiologia , Hepatócitos/fisiologia , Fígado/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Adenilato Quinase/genética , Adenilato Quinase/metabolismo , Animais , Células Cultivadas , Fígado Gorduroso/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Inativação Gênica , Glicogênio/metabolismo , Células Hep G2 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Transcriptoma , Proteína Supressora de Tumor p53/genéticaRESUMO
Adipocyte plasma membrane-associated protein (APMAP) has been described as an adipogenic factor in 3T3-L1 cells with unknown biochemical function; we therefore aimed to investigate the physiologic function of APMAP in vivo We generated Apmap-knockout mice and challenged them with an obesogenic diet to investigate their metabolic phenotype. We identified a novel truncated adipocyte-specific isoform of APMAP in mice that is produced by alternative transcription. Mice lacking the full-length APMAP protein, the only isoform that is expressed in humans, have an improved metabolic phenotype upon diet-induced obesity, indicated by enhanced insulin sensitivity, preserved glucose tolerance, increased respiratory exchange ratio, decreased inflammatory marker gene expression, and reduced adipocyte size. At the molecular level, APMAP interacts with the extracellular collagen cross-linking matrix proteins lysyl oxidase-like 1 and 3. On a high-fat diet, the expression of lysyl oxidase-like 1 and 3 is strongly decreased in Apmap-knockout mice, paralleled by reduced expression of profibrotic collagens and total collagen content in epididymal white adipose tissue, indicating decreased fibrotic potential. Together, our data suggest that APMAP is a novel regulator of extracellular matrix components, and establish that APMAP is a potential target to mitigate obesity-associated insulin resistance.-Pessentheiner, A. R., Huber, K., Pelzmann, H. J., Prokesch, A., Radner, F. P. W., Wolinski, H., Lindroos-Christensen, J., Hoefler, G., Rülicke, T., Birner-Gruenberger, R., Bilban, M., Bogner-Strauss, J. G. APMAP interacts with lysyl oxidase-like proteins, and disruption of Apmap leads to beneficial visceral adipose tissue expansion.
Assuntos
Aminoácido Oxirredutases/metabolismo , Regulação da Expressão Gênica/fisiologia , Gordura Intra-Abdominal/metabolismo , Glicoproteínas de Membrana/metabolismo , Adipócitos/citologia , Adipócitos/fisiologia , Aminoácido Oxirredutases/genética , Animais , Tamanho Celular , Dieta Hiperlipídica , Regulação para Baixo , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Obesidade , Isoformas de ProteínasRESUMO
The importance of peroxisomes for adipocyte function is poorly understood. Herein, we provide insights into the critical role of peroxin 16 (PEX16)-mediated peroxisome biogenesis in adipocyte development and lipid metabolism. Pex16 is highly expressed in adipose tissues and upregulated during adipogenesis of murine and human cells. We demonstrate that Pex16 is a target gene of the adipogenesis "master-regulator" PPARγ. Stable silencing of Pex16 in 3T3-L1 cells strongly reduced the number of peroxisomes while mitochondrial number was unaffected. Concomitantly, peroxisomal fatty acid (FA) oxidation was reduced, thereby causing accumulation of long- and very long-chain (polyunsaturated) FAs and reduction of odd-chain FAs. Further, Pex16-silencing decreased cellular oxygen consumption and increased FA release. Additionally, silencing of Pex16 impaired adipocyte differentiation, lipogenic and adipogenic marker gene expression, and cellular triglyceride stores. Addition of PPARγ agonist rosiglitazone and peroxisome-related lipid species to Pex16-silenced 3T3-L1 cells rescued adipogenesis. These data provide evidence that PEX16 is required for peroxisome biogenesis and highlights the relevance of peroxisomes for adipogenesis and adipocyte lipid metabolism.
Assuntos
Adipócitos Brancos/metabolismo , Homeostase/fisiologia , Metabolismo dos Lipídeos/fisiologia , Lipídeos/fisiologia , Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo , Células 3T3-L1 , Adipogenia/fisiologia , Animais , Células COS , Diferenciação Celular/fisiologia , Linhagem Celular , Chlorocebus aethiops , Ácidos Graxos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Consumo de Oxigênio/fisiologia , PPAR gama/metabolismo , Regulação para Cima/fisiologiaRESUMO
NAT8L (N-acetyltransferase 8-like) catalyzes the formation of N-acetylaspartate (NAA) from acetyl-CoA and aspartate. In the brain, NAA delivers the acetate moiety for synthesis of acetyl-CoA that is further used for fatty acid generation. However, its function in other tissues remained elusive. Here, we show for the first time that Nat8l is highly expressed in adipose tissues and murine and human adipogenic cell lines and is localized in the mitochondria of brown adipocytes. Stable overexpression of Nat8l in immortalized brown adipogenic cells strongly increases glucose incorporation into neutral lipids, accompanied by increased lipolysis, indicating an accelerated lipid turnover. Additionally, mitochondrial mass and number as well as oxygen consumption are elevated upon Nat8l overexpression. Concordantly, expression levels of brown marker genes, such as Prdm16, Cidea, Pgc1α, Pparα, and particularly UCP1, are markedly elevated in these cells. Treatment with a PPARα antagonist indicates that the increase in UCP1 expression and oxygen consumption is PPARα-dependent. Nat8l knockdown in brown adipocytes has no impact on cellular triglyceride content, lipogenesis, or oxygen consumption, but lipolysis and brown marker gene expression are increased; the latter is also observed in BAT of Nat8l-KO mice. Interestingly, the expression of ATP-citrate lyase is increased in Nat8l-silenced adipocytes and BAT of Nat8l-KO mice, indicating a compensatory mechanism to sustain the acetyl-CoA pool once Nat8l levels are reduced. Taken together, our data show that Nat8l impacts on the brown adipogenic phenotype and suggests the existence of the NAT8L-driven NAA metabolism as a novel pathway to provide cytosolic acetyl-CoA for lipid synthesis in adipocytes.
Assuntos
Acetiltransferases/metabolismo , Adipócitos Marrons/metabolismo , Metabolismo Energético , Metabolismo dos Lipídeos , Acetilcoenzima A/metabolismo , Acetiltransferases/deficiência , Acetiltransferases/genética , Adipócitos Marrons/citologia , Adipócitos Marrons/enzimologia , Adipogenia , Animais , Proteínas de Ciclo Celular/metabolismo , Regulação Enzimológica da Expressão Gênica , Técnicas de Inativação de Genes , Inativação Gênica , Humanos , Canais Iônicos/metabolismo , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Tamanho Mitocondrial , PPAR alfa/metabolismo , Fenótipo , Fosfoproteínas/metabolismo , Proteínas Quinases/genética , Transporte Proteico , Proteína Desacopladora 1 , Regulação para CimaRESUMO
Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme mediating triglyceride (TG) hydrolysis. The lack of ATGL results in TG accumulation in multiple tissues, underscoring the critical role of ATGL in maintaining lipid homeostasis. Recent evidence suggests that ATGL affects TG metabolism via activation of peroxisome proliferator-activated receptor α (PPARα). To investigate specific effects of intestinal ATGL on lipid metabolism we generated mice lacking ATGL exclusively in the intestine (ATGLiKO). We found decreased TG hydrolase activity and increased intracellular TG content in ATGLiKO small intestines. Intragastric administration of [(3)H]trioleate resulted in the accumulation of radioactive TG in the intestine, whereas absorption into the systemic circulation was unchanged. Intraperitoneally injected [(3)H]oleate also accumulated within TG in ATGLiKO intestines, indicating that ATGL mobilizes fatty acids from the systemic circulation absorbed by the basolateral side from the blood. Down-regulation of PPARα target genes suggested modulation of cholesterol absorption by intestinal ATGL. Accordingly, ATGL deficiency in the intestine resulted in delayed cholesterol absorption. Importantly, this study provides evidence that ATGL has no impact on intestinal TG absorption but hydrolyzes TGs taken up from the intestinal lumen and systemic circulation. Our data support the role of ATGL in modulating PPARα-dependent processes also in the small intestine.
Assuntos
Intestino Delgado/citologia , Intestino Delgado/enzimologia , Lipase/metabolismo , PPAR alfa/metabolismo , Transdução de Sinais , Triglicerídeos/metabolismo , Animais , Transporte Biológico , Colesterol/metabolismo , Regulação para Baixo , Ácidos Graxos não Esterificados/metabolismo , Fezes/química , Técnicas de Inativação de Genes , Homeostase , Absorção Intestinal , Intestino Delgado/metabolismo , Lipase/deficiência , Lipase/genética , Masculino , Camundongos , Especificidade de Órgãos , Triglicerídeos/sangueRESUMO
BACKGROUND: Fasting induces specific molecular and metabolic adaptions in most organisms. In biomedical research fasting is used in metabolic studies to synchronize nutritional states of study subjects. Because there is a lack of standardization for this procedure, we need a deeper understanding of the dynamics and the molecular mechanisms in fasting. RESULTS: We investigated the dynamic changes of liver gene expression and serum parameters of mice at several time points during a 48 hour fasting experiment and then focused on the global gene expression changes in epididymal white adipose tissue (WAT) as well as on pathways common to WAT, liver, and skeletal muscle. This approach produced several intriguing insights: (i) rather than a sequential activation of biochemical pathways in fasted liver, as current knowledge dictates, our data indicates a concerted parallel response; (ii) this first characterization of the transcriptome signature of WAT of fasted mice reveals a remarkable activation of components of the transcription apparatus; (iii) most importantly, our bioinformatic analyses indicate p53 as central node in the regulation of fasting in major metabolic tissues; and (iv) forced expression of Ddit4, a fasting-regulated p53 target gene, is sufficient to augment lipolysis in cultured adipocytes. CONCLUSIONS: In summary, this combination of focused and global profiling approaches provides a comprehensive molecular characterization of the processes operating during fasting in mice and suggests a role for p53, and its downstream target Ddit4, as novel components in the transcriptional response to food deprivation.
Assuntos
Fatores de Transcrição/metabolismo , Transcriptoma , Proteína Supressora de Tumor p53/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Linhagem Celular , Privação de Alimentos , Perfilação da Expressão Gênica , Gluconeogênese , Lipogênese , Lipólise , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Anotação de Sequência Molecular , Músculo Esquelético/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais , Estresse Fisiológico , Fatores de Transcrição/genética , Ativação Transcricional , Proteína Supressora de Tumor p53/genética , Regulação para CimaRESUMO
Retrotransposed sequences arise from messenger RNAs (mRNAs) that have been reinserted into genomic DNA by reverse transcription. Usually, these sequences are embedded in dormant regions, collect missense mutations over time and constitute processed, nonfunctional pseudogenes. There are thousands of processed pseudogenes in the mouse and human genome. Here, we report evidence for two paralog genes (termed Arxes1 and Arxes2), which arose by retrotransposition of the signal peptidase Spcs3 followed by a segmental duplication event. They gained a functional promoter that we show to be transactivated by adipogenic transcription factors. We further show that the Arxes mRNAs are highly expressed in adipose tissue and strongly upregulated during adipogenesis in different cell models. Additionally, their expression is elevated by an anti-diabetic agent in vitro and in vivo. Importantly, we provide evidence that the Arxes genes are translated and that the proteins are located in the endoplasmic reticulum. Although the sequence similarity and subcellular location are reminiscent of their parental gene, our data suggest that the Arxes have developed a different function, since their expression is required for adipogenesis, whereas Spcs3 is dispensable. In summary, we report retrotransposed-duplicated genes that evolved from a parental gene to function in a tissue and adipogenesis-specific context.
Assuntos
Adipogenia/genética , Peptídeo Hidrolases/fisiologia , Retroelementos , Células 3T3-L1 , Tecido Adiposo/metabolismo , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Células Cultivadas , Perfilação da Expressão Gênica , Genômica , Hipoglicemiantes/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/citologia , Osteogênese , PPAR gama/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Regulação para CimaRESUMO
Triacylglycerols are stored in eukaryotic cells within lipid droplets (LD). The LD core is enwrapped by a phospholipid monolayer with phosphatidylcholine (PC), the major phospholipid, and phosphatidylethanolamine (PE), a minor component. We demonstrate that the onset of LD formation is characterized by a change in cellular PC, PE, and phosphatidylserine (PS). With induction of differentiation of 3T3-L1 fibroblasts into adipocytes, the cellular PC/PE ratio decreased concomitant with LD formation, with the most pronounced decline between confluency and day 5. The mRNA for PS synthase-1 (forms PS from PC) and PS decarboxylase (forms PE from PS) increased after day 5. Activity and protein of PE N-methyltransferase (PEMT), which produces PC by methylation of PE, are absent in 3T3-L1 fibroblasts but were induced at day 5. High fat challenge induced PEMT expression in mouse adipose tissue. PE, produced via PS decarboxylase, was the preferred substrate for methylation to PC. A PEMT-GFP fusion protein decorated the periphery of LD. PEMT knockdown in 3T3-L1 adipocytes correlated with increased basal triacylglycerol hydrolysis. Pemt(-/-) mice developed desensitization against adenosine-mediated inhibition of basal hydrolysis in adipose tissue, and adipocyte hypotrophy was observed in Pemt(-/-) animals on a high fat diet. Knock-out of PEMT in adipose tissue down-regulated PS synthase-1 mRNA, suggesting coordination between PE supply and converting pathways during LD biosynthesis. We conclude that two consecutive processes not previously related to LD biogenesis, (i) PE production via PS and (ii) PE conversion via PEMT, are implicated in LD formation and stability.
Assuntos
Lipídeos/química , Metilação , Fosfatidiletanolaminas/química , Células 3T3 , Adipócitos/citologia , Tecido Adiposo/metabolismo , Animais , Inativação Gênica , Proteínas de Fluorescência Verde/metabolismo , Hidrólise , Masculino , Camundongos , Camundongos Transgênicos , Fosfatidilcolinas/química , Fosfatidilserinas/químicaRESUMO
Infiltration of monocytes and macrophages into the site of inflammation is critical in the progression of inflammatory diseases such as atherosclerosis. Cell migration is dependent on the continuous organization of the actin cytoskeleton, which is regulated by members of the small Rho GTPase family (RhoA, Cdc42, Rac) that are also important for the regulation of signal transduction pathways. We have recently reported on reduced plaque formation in an atherosclerotic mouse model transplanted with bone marrow from adipose triglyceride lipase-deficient (Atgl-/-) mice. Here we provide evidence that defective lipolysis in macrophages lacking ATGL, the major enzyme responsible for triacylglycerol hydrolysis, favors an anti-inflammatory M2-like macrophage phenotype. Our data implicate an as yet unrecognized principle that insufficient lipolysis influences macrophage polarization and actin polymerization, resulting in impaired macrophage migration. Sustained phosphorylation of focal adhesion kinase [due to inactivation of its phosphatase by elevated levels of reactive oxygen species (ROS)] results in defective Cdc42, Rac1 and RhoA activation and in increased and sustained activation of Rac2. Inhibition of ROS production restores the migratory capacity of Atgl-/- macrophages. Since monocyte and macrophage migration are a prerequisite for infiltrating the arterial wall, our results provide a molecular link between lipolysis and the development of atherosclerosis.
Assuntos
Lipólise , Macrófagos/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Movimento Celular , Polaridade Celular , Feminino , Lipase/deficiência , Lipase/metabolismo , Macrófagos/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismoRESUMO
Adipose triglyceride lipase (ATGL) was recently identified as an important triacylglycerol (TG) hydrolase promoting the catabolism of stored fat in adipose and nonadipose tissues. We now demonstrate that efficient ATGL enzyme activity requires activation by CGI-58. Mutations in the human CGI-58 gene are associated with Chanarin-Dorfman Syndrome (CDS), a rare genetic disease where TG accumulates excessively in multiple tissues. CGI-58 interacts with ATGL, stimulating its TG hydrolase activity up to 20-fold. Alleles of CGI-58 carrying point mutations associated with CDS fail to activate ATGL. Moreover, CGI-58/ATGL coexpression attenuates lipid accumulation in COS-7 cells. Antisense RNA-mediated reduction of CGI-58 expression in 3T3-L1 adipocytes inhibits TG mobilization. Finally, expression of functional CGI-58 in CDS fibroblasts restores lipolysis and reverses the abnormal TG accumulation typical for CDS. These data establish an important biochemical function for CGI-58 in the lipolytic degradation of fat, implicating this lipolysis activator in the pathogenesis of CDS.
Assuntos
Tecido Adiposo/enzimologia , Hidrolases de Éster Carboxílico/metabolismo , Esterases/metabolismo , Lipase/metabolismo , Erros Inatos do Metabolismo Lipídico/metabolismo , Lipólise , Fosfolipases A/metabolismo , Triglicerídeos/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase , Células 3T3-L1 , Animais , Células COS , Hidrolases de Éster Carboxílico/genética , Chlorocebus aethiops , Ativação Enzimática , Esterases/genética , Fibroblastos/enzimologia , Inativação Gênica , Humanos , Lipase/genética , Erros Inatos do Metabolismo Lipídico/genética , Camundongos , Mutação , Fosfolipases A/genética , Síndrome , TransfecçãoRESUMO
Obesity is characterized by excessive adipose tissue mass and associated with type 2 diabetes and cardiovascular diseases. To fight obesity and its sequels, elucidating molecular events that govern adipocyte differentiation and function is of key importance. MicroRNAs (miRNAs) are a novel class of non-coding, regulatory RNAs that have been shown to regulate crucial cellular processes, including differentiation. Several studies have already assigned miRNAs to distinct functions in murine adipocyte differentiation but only a few studies did so for humans. Here, we investigated the function of miR-30c in human adipogenesis. miR-30c expression was increased during adipogenesis of human multipotent adipose-derived stem (hMADS) cells, and miR-30c overexpression enforced adipocyte marker gene induction and triglyceride accumulation. miRNA target prediction revealed two putative direct targets of miR-30c, PAI-1 (SERPINE1) and ALK2 (ACVR1, ACTRI), both inversely regulated to miR-30c during adipogenesis and responsive to miR-30c overexpression. Luciferase reporter assays confirmed PAI-1 and ALK2 as direct miR-30c targets. Moreover, reciprocal expression between miR-30c and PAI-1 could also be demonstrated in white adipose tissue of obesity mouse models, suggesting a potential physiological role of miR-30c for PAI-1 regulation in the obese state. Validating PAI-1 and ALK-2 as miR-30c mediators in adipogenesis revealed that not single silencing of PAI-1 or ALK2, but only co-silencing of both phenocopied the pro-adipogenic miR-30c effect. Thus, miR-30c can target two, so far not interconnected genes in distinct pathways, supporting the idea that miRNAs might coordinate larger regulatory networks than previously anticipated.
Assuntos
Receptores de Ativinas Tipo I/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Adipogenia/genética , MicroRNAs/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Receptores de Ativinas Tipo I/genética , Tecido Adiposo Branco/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Obesos , Camundongos Transgênicos , MicroRNAs/genética , Células-Tronco Multipotentes/metabolismo , Obesidade , Inibidor 1 de Ativador de Plasminogênio/genética , Interferência de RNA , RNA Interferente PequenoRESUMO
We have developed a method for reconstructing gene association networks and have applied this method to gene profiles from 3T3-L1 cells. Priorization of the candidate genes pinpointed a transcript annotated as APMAP (adipocyte plasma membrane-associated protein). Functional studies showed that APMAP is upregulated in murine and human adipogenic cell models as well as in a genetic mouse model of obesity. Silencing APMAP in 3T3-L1 cells strongly impaired the differentiation into adipocytes. Moreover, APMAP expression was strongly induced by the PPARγ ligand rosiglitazone in adipocytes in vitro and in vivo in adipose tissue. Using ChIP-qPCR and luciferase reporter assays, we show a functional PPARγ binding site. In addition, we provide evidence that the extracellular C-terminal domain of APMAP is required for the function of APMAP in adipocyte differentiation. Finally, we demonstrate that APMAP translocates from the endoplasmatic reticulum to the plasma membrane during adipocyte differentiation.
Assuntos
Adipogenia/genética , Redes Reguladoras de Genes , Glicoproteínas de Membrana/metabolismo , PPAR gama/metabolismo , Células 3T3-L1 , Algoritmos , Sequência de Aminoácidos , Animais , Humanos , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , PPAR gama/genética , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de SequênciaRESUMO
Oxidized phospholipids (OxPLs) containing enzymatically or non-enzymatically oxidized fatty acids (oxylipins) are increasingly recognized as lipid mediators involved in pathogenesis of diseases. Further understanding of structure-activity relationship and molecular mechanisms activated by OxPLs is hampered by the complexity of synthesis of individual molecular species. Although dozens of individual free oxylipins are commercially available, their attachment to the phospholipid scaffold requires relatively harsh conditions during activation of carboxy-group, which may lead to decomposition of unstable oxylipins. Furthermore, additional protection-deprotection steps are required for oxylipins containing hydroxy-groups. In this work we describe synthesis of OxPLs containing oxylipins bound at the sn-2-position via an amide-bond that is characteristic of sphingophospholipids. Activation of oxylipins and attachment to the phospholipid scaffold are performed under mild conditions and characterized by high yield. Hydroxy-groups of oxylipins do not interfere with reactions and therefore no protection/deprotection steps are needed. In order to prevent oxylipin migration, a fatty acid residue at the sn-1 was bound through an alkyl bond, which is a common bond present in a large proportion of naturally occurring phospholipids. An additional advantage of combining alkyl and amide bonds in a single phospholipid molecule is that both types of bonds are phospholipase A1/A2-resistant, which may be expected to improve biological stability of OxPLs and thus simplify analysis of their effects. As proof of principle, several alkyl-amide oxidized phosphatidylcholines (OxPCs) containing either linear or prostane ring oxylipins have been synthesized. Importantly, we show here that alkyl-amide-OxPCs demonstrated biological activities similar to those of di-acyl-OxPCs. Alkyl-amide-OxPCs inhibited pro-inflammatory action of LPS and increased endothelial cellular barrier in vitro and in mouse models. The effects of alkyl-amide and di-acyl-OxPCs developed in a similar range of concentrations. We hypothesize that alkyl-amide-OxPLs may become a useful tool for deeper analysis of the structure-activity relationship of OxPLs.
Assuntos
Endotoxinas , Fosfolipídeos , Amidas , Animais , Camundongos , Oxirredução , FosfatidilcolinasRESUMO
BACKGROUND: Aspartate biosynthesis and its delivery to the cytosol can be crucial for tumor growth in vivo. However, the impact of intracellular aspartate levels on metastasis has not been studied. We previously described that loss-of-aspartate glutamate carrier 1 (SLC25A12 or AGC1), an important component of the malate-aspartate shuttle, impairs cytosolic aspartate levels, NAD+/NADH ratio, mitochondrial respiration, and tumor growth. Here, we report the impact of AGC1-knockdown on metastasis. RESULTS: Low AGC1 expression correlates with worse patient prognosis in many cancers. AGC1-knockdown in mouse lung carcinoma and melanoma cell lines leads to increased pulmonary metastasis following subcutaneous or intravenous injections, respectively. On the other hand, conventional in vitro metastasis assays show no indication of increased metastasis capacity of AGC1-knockdown cells. CONCLUSION: This study highlights that certain branches of metabolism impact tumor growth and tumor metastasis differently. In addition, it also argues that commonly known metastasis indicators, including EMT genes, cell migration, or colony formation, do not always reflect metastatic capacity in vivo.
RESUMO
Cancer cells rely on glutamine to fuel mitochondria, however it remains unclear whether this is needed for bioenergetic or biosynthetic pathways. Our study suggests that an essential function of mitochondrial glutamine metabolism is to provide aspartate to the cytosol where it can be used for nucleotide and protein synthesis.
RESUMO
OBJECTIVE: The dynamic regulation of metabolic pathways can be monitored by stable isotope tracing. Yet, many metabolites are part of distinct processes within different subcellular compartments. Standard isotope tracing experiments relying on analyses in whole cells may not accurately reflect compartmentalized metabolic processes. Analysis of compartmentalized metabolism and the dynamic interplay between compartments can potentially be achieved by stable isotope tracing followed by subcellular fractionation. Although it is recognized that metabolism can take place during biochemical fractionation of cells, a clear understanding of how such post-harvest metabolism impacts the interpretation of subcellular isotope tracing data and methods to correct for this are lacking. We set out to directly assess artifactual metabolism, enabling us to develop and test strategies to correct for it. We apply these techniques to examine the compartment-specific metabolic kinetics of 13C-labeled substrates targeting central metabolic pathways. METHODS: We designed a stable isotope tracing strategy to interrogate post-harvest metabolic activity during subcellular fractionation using liquid chromatography-mass spectrometry (LC-MS). RESULTS: We show that post-harvest metabolic activity occurs rapidly (within seconds) upon cell harvest. With further characterization we reveal that this post-harvest metabolism is enzymatic and reflects the metabolic capacity of the sub-cellular compartment analyzed, but it is limited in the extent of its propagation into downstream metabolites in metabolic pathways. We also propose and test a post-labeling strategy to assess the amount of post-harvest metabolism occurring in an experiment and then to adjust data to account for this. We validate this approach for both mitochondrial and cytosolic metabolic analyses. CONCLUSIONS: Our data indicate that isotope tracing coupled with sub-cellular fractionation can reveal distinct and dynamic metabolic features of cellular compartments, and that confidence in such data can be improved by applying a post-labeling correction strategy. We examine compartmentalized metabolism of acetate and glutamine and show that acetyl-CoA is turned over rapidly in the cytosol and acts as a pacemaker of anabolic metabolism in this compartment.