RESUMO
BACKGROUND/AIMS: Compared with non-obese individuals, obese individuals commonly store more vitamin D in adipose tissue. VDR expression in adipose tissue can influence adipogenesis and is therefore a target pathway deserving further study. This study aims to assess the role of 1,25(OH)2D3 in human preadipocyte proliferation and differentiation. METHODS: RTCA, MTT, and trypan blue assays were used to assess the effects of 1,25(OH)2D3 on the viability, proliferation, and adipogenic differentiation of SGBS cells. Cell cycle and apoptosis analyses were performed with flow cytometry, triglycerides were quantified, and RT-qPCR was used to assess gene expression. RESULTS: We confirmed that the SGBS cell model is suitable for studying adipogenesis and demonstrated that the differentiation protocol induces cell maturation, thereby increasing the lipid content of cells independently of treatment. 1,25(OH)2D3 treatment had different effects according to the cell stage, indicating different modes of action driving proliferation and differentiation. In preadipocytes, 1,25(OH)2D3 induced G1 growth arrest at both tested concentrations without altering CDKN1A gene expression. Treatment with 100 nM 1,25(OH)2D3 also decreased MTT absorbance and the lipid concentration. Moreover, increased normalized cell index values and decreased metabolic activity were not induced by proliferation or apoptosis. Exposure to 100 nM 1,25(OH)2D3 induced VDR, CEBPA, and CEBPB expression, even in the preadipocyte stage. During adipogenesis, 1,25(OH)2D3 had limited effects on processes such as VDR and PPARG gene expression, but it upregulated CEBPA expression. CONCLUSIONS: We demonstrated for the first time that 1,25(OH)2D3 induces changes in preadipocytes, including VDR expression and growth arrest, and increases the lipid content in adipocytes treated for 16 days. Preadipocytes are important cells in adipose tissue homeostasis, and understanding the role of 1,25(OH)2D3 in adipogenesis is a crucial step in ensuring adequate vitamin D supplementation, especially for obese individuals.
Assuntos
Adipogenia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Vitamina D/análogos & derivados , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , PPAR gama/genética , PPAR gama/metabolismo , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Regulação para Cima/efeitos dos fármacos , Vitamina D/farmacologiaRESUMO
The analysis of transcriptomics data is able to give an overview of cellular processes, but requires sophisticated bioinformatics tools and methods to identify the changes. Pathway analysis software, like PathVisio, captures the information about biological pathways from databases and brings this together with the experimental data to enable visualization and understanding of the underlying processes. Rett syndrome is a rare disease, but still one of the most abundant causes of intellectual disability in females. Cause of this neurological disorder is mutation of one single gene, the methyl-CpG-binding protein 2 (MECP2) gene. This gene is responsible for many steps in neuronal development and function. Although the genetic mutation and the clinical phenotype are well described, the molecular pathways linking them are not yet fully elucidated. In this study we demonstrate a workflow for the analysis of transcriptomics data to identify biological pathways and processes which are changed in a Mecp2 (-/y) mouse model.
Assuntos
Perfilação da Expressão Gênica , Síndrome de Rett/diagnóstico , Síndrome de Rett/genética , Animais , Criança , Pré-Escolar , Biologia Computacional , Modelos Animais de Doenças , Feminino , Humanos , Lactente , Recém-Nascido , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , FenótipoRESUMO
RATIONALE: Viral myocarditis results from an adverse immune response to cardiotropic viruses, which causes irreversible myocyte destruction and heart failure in previously healthy people. The involvement of microRNAs and their usefulness as therapeutic targets in this process are unknown. OBJECTIVE: To identify microRNAs involved in viral myocarditis pathogenesis and susceptibility. METHODS AND RESULTS: Cardiac microRNAs were profiled in both human myocarditis and in Coxsackievirus B3-injected mice, comparing myocarditis-susceptible with nonsusceptible mouse strains longitudinally. MicroRNA responses diverged depending on the susceptibility to myocarditis after viral infection in mice. MicroRNA-155, -146b, and -21 were consistently and strongly upregulated during acute myocarditis in both humans and susceptible mice. We found that microRNA-155 expression during myocarditis was localized primarily in infiltrating macrophages and T lymphocytes. Inhibition of microRNA-155 by a systemically delivered LNA-anti-miR attenuated cardiac infiltration by monocyte-macrophages, decreased T lymphocyte activation, and reduced myocardial damage during acute myocarditis in mice. These changes were accompanied by the derepression of the direct microRNA-155 target PU.1 in cardiac inflammatory cells. Beyond the acute phase, microRNA-155 inhibition reduced mortality and improved cardiac function during 7 weeks of follow-up. CONCLUSIONS: Our data show that cardiac microRNA dysregulation is a characteristic of both human and mouse viral myocarditis. The inflammatory microRNA-155 is upregulated during acute myocarditis, contributes to the adverse inflammatory response to viral infection of the heart, and is a potential therapeutic target for viral myocarditis.
Assuntos
Infecções por Coxsackievirus/genética , Perfilação da Expressão Gênica , MicroRNAs/metabolismo , Miocardite/genética , Miocárdio/metabolismo , Animais , Infecções por Coxsackievirus/imunologia , Infecções por Coxsackievirus/patologia , Infecções por Coxsackievirus/fisiopatologia , Infecções por Coxsackievirus/terapia , Infecções por Coxsackievirus/virologia , Modelos Animais de Doenças , Enterovirus Humano B/patogenicidade , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Ativação Linfocitária , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Miocardite/imunologia , Miocardite/patologia , Miocardite/fisiopatologia , Miocardite/terapia , Miocardite/virologia , Miocárdio/imunologia , Miocárdio/patologia , Oligonucleotídeos/administração & dosagem , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/virologia , Fatores de TempoRESUMO
Although CPT-I (carnitine palmitoyltransferase-I) is generally regarded to present a major rate-controlling site in mitochondrial beta-oxidation, it is incompletely understood whether CPT-I is rate-limiting in the overall LCFA (long-chain fatty acid) flux in the heart. Another important site of regulation of the LCFA flux in the heart is trans-sarcolemmal LCFA transport facilitated by CD36 and FABPpm (plasma membrane fatty acid-binding protein). Therefore, we explored to what extent a chronic pharmacological blockade of the LCFA flux at the level of mitochondrial entry of LCFA-CoA would affect sarcolemmal LCFA uptake. Rats were injected daily with saline or etomoxir, a specific CPT-I inhibitor, for 8 days at 20 mg/kg of body mass. Etomoxir-treated rats displayed a 44% reduced cardiac CPT-I activity. Sarcolemmal contents of CD36 and FABPpm, as well as the LCFA transport capacity, were not altered in the hearts of etomoxir-treated versus control rats. Furthermore, rates of LCFA uptake and oxidation, and glucose uptake by cardiac myocytes from etomoxir-treated rats were not different from control rats, neither under basal nor under acutely induced maximal metabolic demands. Finally, hearts from etomoxir-treated rats did not display triacylglycerol accumulation. Therefore CPT-I appears not to present a major rate-controlling site in total cardiac LCFA flux. It is likely that sarcolemmal LCFA entry rather than mitochondrial LCFA-CoA entry is a promising target for normalizing LCFA flux in cardiac metabolic diseases.
Assuntos
Carnitina O-Palmitoiltransferase/metabolismo , Compostos de Epóxi/farmacologia , Ácidos Graxos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Western Blotting , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Masculino , Oxirredução/efeitos dos fármacos , Ratos , Triglicerídeos/metabolismoRESUMO
Contraction-induced glucose uptake is only partly mediated by AMPK activation. We examined whether the diacylglycerol-sensitive protein kinase D (PKD; also known as novel PKC isoform mu) is also involved in the regulation of glucose uptake in the contracting heart. As an experimental model, we used suspensions of cardiac myocytes, which were electrically stimulated to contract or treated with the contraction-mimicking agent oligomycin. Induction of contraction at 4 Hz in cardiac myocytes or treatment with 1 microM oligomycin enhanced (i) autophosphorylation of PKD at Ser916 by 5.1- and 3.8-fold, respectively, (ii) phosphorylation of PKD's downstream target cardiac-troponin-I (cTnI) by 2.9- and 2.1-fold, respectively, and (iii) enzymatic activity of immunoprecipitated PKD towards the substrate peptide syntide-2 each by 1.5-fold. Although AMPK was also activated under these same conditions, in vitro phosphorylation assays and studies with cardiac myocytes from AMPKalpha2(-/-) mice indicated that activation of PKD occurs independent of AMPK activation. CaMKKbeta, and the cardiac-specific PKC isoforms alpha, delta, and epsilon were excluded as upstream kinases for PKD in contraction signaling because none of these kinases were activated by oligomycin. Stimulation of glucose uptake and induction of GLUT4 translocation in cardiac myocytes by contraction and oligomycin each were sensitive to inhibition by the PKC/PKD inhibitors staurosporin and calphostin-C. Together, these data elude to a role of PKD in contraction-induced GLUT4 translocation. Finally, the combined actions of PKD on cTnI phosphorylation and on GLUT4 translocation would efficiently link accelerated contraction mechanics to increased energy production when the heart is forced to increase its contractile activity.
Assuntos
Transportador de Glucose Tipo 4/metabolismo , Glucose/metabolismo , Complexos Multienzimáticos/metabolismo , Contração Muscular , Miócitos Cardíacos/metabolismo , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas Quinases Ativadas por AMP , Animais , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Desoxiglucose/metabolismo , Estimulação Elétrica , Ativação Enzimática , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Camundongos , Camundongos Knockout , Complexos Multienzimáticos/deficiência , Complexos Multienzimáticos/genética , Contração Muscular/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Naftalenos/farmacologia , Oligomicinas/farmacologia , Peptídeos/metabolismo , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Estaurosporina/farmacologia , Troponina I/metabolismoRESUMO
Long-term weight loss maintenance is a problem of overweight and obesity. Changes of gene expression during weight loss (WL) by calorie restriction (CR) are linked to the risk of weight regain (WR). However, detailed information on genes/proteins involved in the mechanism is still lacking. Therefore, we developed an in-vitro model system for glucose restriction (GR) and refeeding (RF) to uncover proteome differences between GR with RF vs normal feeding, of which we explored the relation with WR after WL. Human Simpson-Golabi-Behmel Syndrome cells were subjected to changing levels of glucose to mimic the condition of CR and RF. Proteome profiling was performed by liquid chromatography tandem mass spectrometry. This in-vitro model revealed 44 proteins differentially expressed after GR and RF versus feeding including proteins of the focal adhesions. Four proteins showed a persistent up- or down-regulation: liver carboxylesterase (CES1), mitochondrial superoxide dismutase [Mn] (SOD2), alpha-crystallin B-chain (CRYAB), alpha-enolase (ENO1). In-vivo weight loss-induced RNA expression changes linked CES1, CRYAB and ENO1 to WR. Moreover, of these 44 proteins, CES1 and glucosidase II alpha subunit (GANAB) during follow up correlated with WR. Correlation clustering of in-vivo protein expression data indicated an interaction of these proteins with structural components of the focal adhesions and cytoplasmic filaments in the adipocytes.
Assuntos
Adipócitos/metabolismo , Biomarcadores Tumorais/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Glucose/deficiência , Glucosidases/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Aumento de Peso , Cadeia B de alfa-Cristalina/metabolismo , Adipócitos/citologia , Biomarcadores Tumorais/genética , Hidrolases de Éster Carboxílico/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Glucose/metabolismo , Glucosidases/genética , Humanos , Fosfopiruvato Hidratase/genética , Proteínas Supressoras de Tumor/genética , Cadeia B de alfa-Cristalina/genéticaRESUMO
Biological pathways are abstract and functional visual representations of existing biological knowledge. By mapping high-throughput data on these representations, changes and patterns in biological systems on the genetic, metabolic and protein level are instantly assessable. Many public domain repositories exist for storing biological pathways, each applying its own conventions and storage format. A pathway-based content review of these repositories reveals that none of them are comprehensive. To address this issue, we apply a general workflow to create curated biological pathways, in which we combine three content sources: public domain databases, literature and experts. In this workflow all content of a particular biological pathway is manually retrieved from biological pathway databases and literature, after which this content is compared, combined and subsequently curated by experts. From the curated content, new biological pathways can be created for a pathway analysis tool of choice and distributed among its user base. We applied this procedure to construct high-quality curated biological pathways involved in human fatty acid metabolism.
Assuntos
Inteligência Artificial , Disciplinas das Ciências Biológicas/normas , Disciplinas das Ciências Biológicas/tendências , Animais , Bases de Dados Factuais , Ácidos Graxos/metabolismo , HumanosRESUMO
Rett syndrome (RTT) is a rare disease but still one of the most abundant causes for intellectual disability in females. Typical symptoms are onset at month 6-18 after normal pre- and postnatal development, loss of acquired skills and severe intellectual disability. The type and severity of symptoms are individually highly different. A single mutation in one gene, coding for methyl-CpG-binding protein 2 (MECP2), is responsible for the disease. The most important action of MECP2 is regulating epigenetic imprinting and chromatin condensation, but MECP2 influences many different biological pathways on multiple levels although the molecular pathways from gene to phenotype are currently not fully understood. In this review the known changes in metabolite levels, gene expression and biological pathways in RTT are summarized, discussed how they are leading to some characteristic RTT phenotypes and therefore the gaps of knowledge are identified. Namely, which phenotypes have currently no mechanistic explanation leading back to MECP2 related pathways? As a result of this review the visualization of the biologic pathways showing MECP2 up- and downstream regulation was developed and published on WikiPathways which will serve as template for future omics data driven research. This pathway driven approach may serve as a use case for other rare diseases, too.
Assuntos
Proteína 2 de Ligação a Metil-CpG/metabolismo , Síndrome de Rett/metabolismo , Síndrome de Rett/patologia , Animais , Biologia Computacional , Metilação de DNA/genética , Modelos Animais de Doenças , Epigenômica , Humanos , Proteína 2 de Ligação a Metil-CpG/genética , Síndrome de Rett/genética , Biologia de SistemasRESUMO
Contraction of rat cardiac myocytes induces translocation of fatty acid translocase (FAT)/CD36 and GLUT4 from intracellular stores to the sarcolemma, leading to enhanced rates of long-chain fatty acid (FA) and glucose uptake, respectively. Because intracellular AMP/ATP is elevated in contracting cardiac myocytes, we investigated whether activation of AMP-activated protein kinase (AMP kinase) is involved in contraction-inducible FAT/CD36 translocation. The cell-permeable adenosine analog 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and the mitochondrial inhibitor oligomycin, similar to 4-Hz electrostimulation, evoked a more than threefold activation of cardiomyocytic AMP kinase. Both AICAR and oligomycin stimulated FA uptake into noncontracting myocytes by 1.4- and 2.0-fold, respectively, but were ineffective in 4 Hz-contracting myocytes. These findings indicate that both agents stimulate FA uptake by a similar mechanism as electrostimulation, involving activation of AMP kinase, as evidenced from phosphorylation of acetyl-CoA carboxylase. Furthermore, the stimulating effects of both AICAR and oligomycin were antagonized by blocking FAT/CD36 with sulfo-N-succinimidylpalmitate, but not by inhibiting phosphatidylinositol 3-kinase with wortmannin, indicating the involvement of FAT/CD36, but excluding a role for insulin signaling. Subcellular fractionation showed that oligomycin was able to mobilize intracellularly stored FAT/CD36 to the sarcolemma. We conclude that AMP kinase regulates cardiac FA use through mobilization of FAT/CD36 from a contraction-inducible intracellular storage compartment.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Coração/fisiologia , Glicoproteínas de Membrana/metabolismo , Complexos Multienzimáticos/metabolismo , Contração Miocárdica/fisiologia , Transportadores de Ânions Orgânicos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Trifosfato de Adenosina/metabolismo , Aminoimidazol Carboxamida/farmacologia , Animais , Antígenos CD36/metabolismo , Desoxiglucose/metabolismo , Estimulação Elétrica , Ácidos Graxos não Esterificados/metabolismo , Coração/efeitos dos fármacos , Técnicas In Vitro , Insulina/farmacologia , Cinética , Masculino , Mitocôndrias Cardíacas/metabolismo , Miocárdio/enzimologia , Miocárdio/metabolismo , Oligomicinas/farmacologia , Ratos , Ratos Endogâmicos Lew , Ribonucleotídeos/farmacologia , Transdução de SinaisRESUMO
In obesity, the development of cardiomyopathy is associated with the accumulation of myocardial triacylglycerols (TAGs), possibly stemming from elevation of myocardial long-chain fatty acid (LCFA) uptake. Because LCFA uptake is regulated by insulin and contractions, we examined in cardiac myocytes from lean and obese Zucker rats the effects of insulin and the contraction-mimetic agent oligomycin on the initial rate of LCFA uptake, subcellular distribution of FAT/CD36, and LCFA metabolism. In cardiac myocytes from obese Zucker rats, under basal conditions, FAT/CD36 was relocated to the sarcolemma at the expense of intracellular stores. In addition, the LCFA uptake rate, LCFA esterification rate into TAGs, and the intracellular unesterified LCFA concentration each were significantly increased. All these metabolic processes were normalized by the FAT/CD36 inhibitor sulfo-N-succinimidyloleate, indicating its antidiabetic potential. In cardiac myocytes isolated from lean rats, in vitro administration of insulin induced the translocation of FAT/CD36 to the sarcolemma and stimulated initial rates of LCFA uptake and TAG esterification. In contrast, in myocytes from obese rats, insulin failed to alter the subcellular localization of FAT/CD36 and the rates of LCFA uptake and TAG esterification. In cardiac myocytes from lean and obese animals, oligomycin stimulated the initial rates of LCFA uptake and oxidation, although oligomycin only induced the translocation of FAT/CD36 to the sarcolemma in lean rats. The present results indicate that in cardiac myocytes from obese Zucker rats, a permanent relocation of FAT/CD36 to the sarcolemma is responsible for myocardial TAG accumulation. Furthermore, in vitro these cardiac myocytes, although sensitive to contraction-like stimulation, were completely insensitive to insulin, as the basal conditions in hyperinsulinemic, obese animals resemble the insulin-stimulated condition in lean littermates.
Assuntos
Antígenos CD36/metabolismo , Miócitos Cardíacos/metabolismo , Obesidade/metabolismo , Sarcolema/metabolismo , Triglicerídeos/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Antígenos CD36/efeitos dos fármacos , Esterificação , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Feminino , Membranas Intracelulares/metabolismo , Miocárdio/metabolismo , Obesidade/patologia , Ácidos Oleicos/farmacologia , Oligomicinas/farmacologia , Oxirredução , Fosfolipídeos/biossíntese , Ratos , Ratos Zucker , Frações Subcelulares/metabolismo , Succinimidas/farmacologia , Magreza/metabolismo , Magreza/patologia , Distribuição TecidualRESUMO
Cellular fatty acid uptake is facilitated by a number of fatty acid transporters, FAT/CD36, FABPpm and FATP1. It had been presumed that FABPpm, was confined to the plasma membrane and was not regulated. Here, we demonstrate for the first time that FABPpm and FATP1 are also present in intracellular depots in cardiac myocytes. While we confirmed previous work that insulin and AICAR each induced the translocation of FAT/CD36 from an intracellular depot to the PM, only AICAR, but not insulin, induced the translocation of FABPpm. Moreover, neither insulin nor AICAR induced the translocation of FATP1. Importantly, the increased plasmalemmal content of these LCFA transporters was associated with a concomitant increase in the initial rate of palmitate uptake into cardiac myocytes. Specifically, the insulin-stimulated increase in the rate of palmitate uptake (+60%) paralleled the insulin-stimulated increase in plasmalemmal FAT/CD36 (+34%). Similarly, the greater AICAR-stimulated increase in the rate of palmitate uptake (+90%) paralleled the AICAR-induced increase in both plasmalemmal proteins (FAT/CD36 (+40%)+FABPpm (+36%)). Inhibition of palmitate uptake with the specific FAT/CD36 inhibitor SSO indicated that FABPpm interacts with FAT/CD36 at the plasma membrane to facilitate the uptake of palmitate. In conclusion, (1) there appears to be tissue-specific sensitivity to insulin-induced FATP1 translocation, as it has been shown elsewhere that insulin induces FATP1 translocation in 3T3-L1 adipocytes, and (2) clearly, the subcellular distribution of FABPpm, as well as FAT/CD36, is acutely regulated in cardiac myocytes, although FABPpm and FAT/CD36 do not necessarily respond identically to the same stimuli.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Antígenos CD36/metabolismo , Proteínas de Transporte/metabolismo , Insulina/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Ribonucleotídeos/farmacologia , Animais , Membrana Celular/metabolismo , Células Cultivadas , Proteínas de Transporte de Ácido Graxo , Proteínas de Ligação a Ácido Graxo , Ácidos Graxos/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Transporte Proteico , RatosRESUMO
In contrast to UCP1, the primary function of UCP3 is not the dissipation of energy. Rather, several lines of evidence suggest that UCP3 is related to cellular long-chain fatty acid homeostasis. If long-chain fatty acids enter the mitochondrial matrix in their non-esterified form, they cannot be metabolized and may exert deleterious effects. To test the feasibility that UCP3 exports fatty acid anions, we systematically interfered at distinct steps in the fatty acid metabolism pathway, thereby creating conditions in which the entry of (non-esterified) fatty acids into the mitochondrial matrix is enhanced. First, reducing the cellular fatty acid binding capacity, known to increase cytosolic concentrations of non-esterified fatty acids, up-regulated UCP3 5.3-fold. Second, inhibition of mitochondrial entry of esterified long-chain fatty acids up-regulated UCP3 by 1.9-fold. Third, high-fat diets, to increase mitochondrial supply of non-esterified long-chain fatty acids exceeding oxidative capacity, up-regulated UCP3 twofold. However, feeding a similar amount of medium-chain fatty acids, which can be oxidized inside the mitochondrial matrix and therefore do not need to be exported from the matrix, did not affect UCP3 protein levels. These data are compatible with a physiological function of UCP3 in facilitating outward transport of long-chain fatty acid anions, which cannot be oxidized, from the mitochondrial matrix.
Assuntos
Proteínas de Transporte/fisiologia , Ácidos Graxos/metabolismo , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Administração Oral , Animais , Ânions/metabolismo , Transporte Biológico , Carnitina O-Palmitoiltransferase/metabolismo , Proteínas de Transporte/genética , Inibidores Enzimáticos/farmacologia , Compostos de Epóxi/farmacologia , Gorduras/administração & dosagem , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Ácidos Graxos/química , Canais Iônicos , Masculino , Camundongos , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Modelos Biológicos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Ratos , Ratos Wistar , Proteína Desacopladora 3RESUMO
BACKGROUND: Proteomic technologies applied for profiling human biofluids and blood cells are considered to reveal new biomarkers of exposure or provide insights into novel mechanisms of adaptation. METHODS: Both a non-targeted (classical 2D-electrophoresis combined with mass spectrometry) as well as a targeted proteomic approach (multiplex immunoassay) were applied to investigate how fasting for 36 h, as compared to 12 h, affects the proteome of platelets, peripheral blood mononuclear cells (PBMC), plasma, urine and saliva collected from ten healthy volunteers. RESULTS: Between-subject variability was highest in the plasma proteome and lowest in the PBMC proteome. Random Forests analysis performed on the entire dataset revealed that changes in the level of the RhoGDI2 protein in PBMC and plasma ApoA4 levels were the two most obvious biomarkers of an extended fasting. Random Forests (RF) analysis of the multiplex immunoassay data revealed leptin and MMP-3 as biomarkers for extended fasting. However, high between-subject variability may have masked the extended fasting effects in the proteome of the biofluids and blood cells. CONCLUSIONS: Identification of significantly changed proteins in biofluids and blood cells using a non-targeted approach, together with the outcome of targeted analysis revealed both known and novel markers for a 36 h fasting period, including the cellular proteins RhoGDI2 and CLIC1, and plasma proteins ApoA4, leptin and MMP-3. The PBMC proteome exhibited the lowest between-subject variability and therefore these cells appear to represent the best biosamples for biomarker discovery in human nutrigenomics.
Assuntos
Células Sanguíneas/metabolismo , Líquidos Corporais/metabolismo , Jejum , Proteoma/metabolismo , Proteômica/métodos , Apolipoproteínas A/sangue , Biomarcadores/metabolismo , Análise por Conglomerados , Eletroforese em Gel Bidimensional , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Humanos , Imunoensaio , Leptina/metabolismo , Espectrometria de Massas , Metaloproteinase 3 da Matriz/metabolismo , Análise de Componente Principal , Fatores de Tempo , Proteínas Supressoras de Tumor/metabolismo , Inibidor beta de Dissociação do Nucleotídeo Guanina rho , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
Insulin resistance is a characteristic of type-2 diabetes and its development is associated with an increased fat consumption. Muscle is one of the tissues that becomes insulin resistant after high fat (HF) feeding. The aim of the present study is to identify processes involved in the development of HF-induced insulin resistance in muscle of ApOE3*Leiden mice by using microarrays. These mice are known to become insulin resistant on a HF diet. Differential gene expression was measured in muscle using the Affymetrix mouse plus 2.0 array. To get more insight in the processes, affected pathway analysis was performed with a new tool, PathVisio. PathVisio is a pathway editor customized with plug-ins (1) to visualize microarray data on pathways and (2) to perform statistical analysis to select pathways of interest. The present study demonstrated that with pathway analysis, using PathVisio, a large variety of processes can be investigated. The significantly regulated genes in muscle of ApOE3*Leiden mice after 12 weeks of HF feeding were involved in several biological pathways including fatty acid beta oxidation, fatty acid biosynthesis, insulin signaling, oxidative stress and inflammation.
RESUMO
Cardiovascular disease is the primary cause of death in obesity and type-2 diabetes mellitus (T2DM). Alterations in substrate metabolism are believed to be involved in the development of both cardiac dysfunction and insulin resistance in these conditions. Under physiological circumstances the heart utilizes predominantly long-chain fatty acids (LCFAs) (60-70%), with the remainder covered by carbohydrates, i.e., glucose (20%) and lactate (10%). The cellular uptake of both LCFA and glucose is regulated by the sarcolemmal amount of specific transport proteins, i.e., fatty acid translocase (FAT)/CD36 and GLUT4, respectively. These transport proteins are not only present at the sarcolemma, but also in intracellular storage compartments. Both an increased workload and the hormone insulin induce translocation of FAT/CD36 and GLUT4 to the sarcolemma. In this review, recent findings on the insulin and contraction signalling pathways involved in substrate uptake and utilization by cardiac myocytes under physiological conditions are discussed. New insights in alterations in substrate uptake and utilization during insulin resistance and its progression towards T2DM suggest a pivotal role for substrate transporters. During the development of obesity towards T2DM alterations in cardiac lipid homeostasis were found to precede alterations in glucose homeostasis. In the early stages of T2DM, relocation of FAT/CD36 to the sarcolemma is associated with the myocardial accumulation of triacylglycerols (TAGs) eventually leading to an impaired insulin-stimulated GLUT4-translocation. These novel insights may result in new strategies for the prevention of development of cardiac dysfunction and insulin resistance in obesity and T2DM.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Miocárdio/metabolismo , Obesidade/metabolismo , Sarcolema/metabolismo , Animais , Ácidos Graxos/metabolismo , Glucose/metabolismo , Humanos , Insulina/metabolismo , Resistência à Insulina , Transdução de SinaisRESUMO
Recently, fatty acid transport across the plasma membrane has been shown to be a key process that contributes to the regulation of fatty acid metabolism in the heart. Since AMP kinase activation by 5-aminoimidazole-4-carboxamide-1-beta-D: -ribofuranoside (AICAR) stimulates fatty acid oxidation, as well as the expression of selected proteins involved with energy provision, we examined (a) whether AICAR induced the expression of the fatty acid transporters FABPpm and FAT/CD36 in cardiac myocytes and in perfused hearts and (b) the signaling pathway involved. Incubation of cardiac myocytes with AICAR increased the protein expression of the fatty acid transporter FABPpm after 90 min (+27%, P < 0.05) and this protein remained stably overexpressed until 180 min. Similarly, FAT/CD36 protein expression was increased after 60 min (+38%, P < 0.05) and remained overexpressed thereafter. Protein overexpression, which occurred via transcriptional mechanisms, was dependent on the AICAR concentration, with optimal induction occurring at AICAR concentrations 1-5 mM for FABPpm and at 2-8 mM for FAT/CD36. The AICAR (2 h, 2 mM AICAR) effects on FABPpm and FAT/CD36 protein expression were similar in perfused hearts and in cardiac myocytes. AICAR also induced the plasmalemmal content of FAT/CD36 (+49%) and FABPpm (+42%) (P < 0.05). This was accompanied by a marked increase in the rate of palmitate transport (2.5 fold) into giant sarcolemmal vesicles, as well as by increased rates of palmitate oxidation in cardiac myocytes. When the AICAR-induced AMPK phosphorylation was blocked, neither FAT/CD36 nor FABPpm were overexpressed, nor were palmitate uptake and oxidation increased. This study has revealed that AMPK activation stimulates the protein expression of both fatty acid transporters, FAT/CD36 and FABPpm in (a) time- and (b) dose-dependent manner via (c) the AMPK signaling pathway. AICAR also (d) increased the plasmalemmal content of FAT/CD36 and FABPm, thereby (e) increasing the rates of fatty acid transport. Thus, activation of AMPK is a key mechanism regulating the expression as well as the plasmalemmal localization of fatty acid transporters.
Assuntos
Antígenos CD36/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Complexos Multienzimáticos/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Relação Dose-Resposta a Droga , Ácidos Graxos/metabolismo , Hipoglicemiantes/farmacologia , Masculino , Miocárdio/enzimologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Oxirredução , Ratos , Ratos Wistar , Ribonucleotídeos/farmacologia , Transdução de SinaisRESUMO
Glucose and long-chain fatty acids (LCFA) are two major substrates used by heart and skeletal muscle to support contractile activity. In quiescent cardiac myocytes a substantial portion of the glucose transporter GLUT4 and the putative LCFA transporter fatty acid translocase (FAT)/CD36 are stored in intracellular compartments. Induction of cellular contraction by electrical stimulation results in enhanced uptake of both glucose and LCFA through translocation of GLUT4 and FAT/CD36 respectively to the sarcolemma. The involvement of protein kinase A, AMP-activated protein kinase (AMPK), protein kinase C (PKC) isoforms and the extracellular signal-regulated kinases was evaluated in cardiac myocytes as candidate signalling enzymes involved in recruiting these transporters in response to contraction. The collected evidence excluded the involvement of PKA and implicated an important role for AMPK and for one (or more) PKC isoform(s) in contraction-induced translocation of both GLUT4 and FAT/CD36. The unravelling of further components along this contraction pathway can provide valuable information on the coordinated regulation of the uptake of glucose and of LCFA by an increase in mechanical activity of heart and skeletal muscle.
Assuntos
Antígenos CD36/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Contração Muscular/fisiologia , Proteínas Musculares/metabolismo , Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , Ácidos Graxos/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 4 , Humanos , Miócitos Cardíacos/enzimologia , Isoformas de Proteínas , Proteínas Quinases/metabolismo , Transdução de SinaisRESUMO
Because insulin has been shown to stimulate long-chain fatty acid (LCFA) esterification in skeletal muscle and cardiac myocytes, we investigated whether insulin increased the rate of LCFA transport by altering the expression and the subcellular distribution of the fatty acid transporters FAT/CD36 and FABPpm. In cardiac myocytes, insulin very rapidly increased the expression of FAT/CD36 protein in a time- and dose-dependent manner. During a 2-h period, insulin (10 nM) increased cardiac myocyte FAT/CD36 protein by 25% after 60 min and attained a maximum after 90-120 min (+40-50%). There was a dose-dependent relationship between insulin (10(-12) to 10(-7) M) and FAT/CD36 expression. The half-maximal increase in FAT/CD36 protein occurred at 0.5 x 10(-9) M insulin, and the maximal increase occurred at 10(-9) to 10(-8) M insulin (+40-50%). There were similar insulin-induced increments in FAT/CD36 protein in cardiac myocytes (+43%) and in Langendorff-perfused hearts (+32%). In contrast to FAT/CD36, insulin did not alter the expression of FABPpm protein in either cardiac myocytes or the perfused heart. By use of specific inhibitors of insulin-signaling pathways, it was shown that insulin-induced expression of FAT/CD36 occurred via the PI 3-kinase/Akt insulin-signaling pathway. Subcellular fractionation of cardiac myocytes revealed that insulin not only increased the expression of FAT/CD36, but this hormone also targeted some of the FAT/CD36 to the plasma membrane while concomitantly lowering the intracellular depot of FAT/CD36. At the functional level, the insulin-induced increase in FAT/CD36 protein resulted in an increased rate of palmitate transport into giant vesicles (+34%), which paralleled the increase in plasmalemmal FAT/CD36 (+29%). The present studies have shown that insulin regulates protein expression of FAT/CD36, but not FABPpm, via the PI 3-kinase/Akt insulin-signaling pathway.
Assuntos
Antígenos CD36/metabolismo , Proteínas de Transporte/metabolismo , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Transportadores de Ânions Orgânicos/metabolismo , Animais , Transporte Biológico Ativo , Northern Blotting , Western Blotting , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação a Ácido Graxo , Técnicas In Vitro , Cinética , Masculino , Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Estimulação Química , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Vesículas Transportadoras/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
In cardiac myocytes, uptake rates of glucose and long-chain fatty acids (FA) are regulated by translocation of GLUT4 and FA translocase (FAT)/CD36, respectively, from intracellular stores to the sarcolemma. Insulin and contractions are two major physiological stimuli able to induce translocation of both transporters and therefore enhance the uptake of both substrates. Interestingly, the cardiovascular drug dipyridamole was able to enhance FA uptake but had no effect on glucose uptake. The selective stimulatory effect of dipyridamole on FA uptake was unrelated to its effects on phosphodiesterase inhibition and on nucleoside transport inhibition. However, dipyridamole-stimulated FA uptake was abolished in the presence of sulfo-N-succinimidylpalmitate, which indicated that FAT/CD36 is involved in the uptake process. Furthermore, the effect was additive to that of insulin but not to that of the AMP-elevating agent oligomycin, indicating that dipyridamole stimulates FAT/CD36-mediated FA uptake by activating the AMP-activated protein kinase (AMPK) signaling pathway. Dipyridamole, however, neither influenced the intracellular AMP content nor induced activation of AMPK. Finally, dipyridamole was able to induce FAT/CD36 translocation from intracellular storage sites to the sarcolemma but had no effect on the subcellular distribution of GLUT4. It is concluded that beyond AMP-activated protein kinase the contraction-induced and AMPK-mediated signal branches off into separate mobilization of GLUT4 and of FAT/CD36, and that dipyridamole activates a yet unidentified target in the FAT/CD36 mobilizing branch.
Assuntos
Antígenos CD36/metabolismo , Dipiridamol/farmacologia , Coração/efeitos dos fármacos , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Miocárdio/metabolismo , Proteínas Quinases Ativadas por AMP , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Ativação Enzimática , Transportador de Glucose Tipo 4 , Masculino , Complexos Multienzimáticos/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Endogâmicos LewRESUMO
Sulfo-N-succinimidyl esters of LCFAs are a powerful tool to investigate the functional significance of plasmalemmal proteins in the LCFA uptake process. This notion is based on the following observations. First, sulfo-N-succinimidyl oleate (SSO) was found to inhibit the bulk of LCFA uptake into various cell types, i.e. rat adipocytes, type II pneumocytes and cardiac myocytes. Second, using cardiac giant membrane vesicles, in which LCFA uptake can be investigated in the absence of mitochondrial beta-oxidation, SSO retained the ability to largely inhibit LCFA uptake, indicating that inhibition of LCFA transsarcolemmal transport is its primary action. Third, SSO has no inhibitory effect on glucose and octanoate uptake into giant membrane vesicles derived from heart and skeletal muscle, indicating that its action is specific for LCFA uptake. Finally, SSO specifically binds to the 88 kDa plasmalemmal fatty acid transporter FAT, a rat homologue of human CD36, resulting in an arrest of the transport function of this protein. In addition to its inhibitory action at the plasma membrane level, evidence is presented for the lack of a direct inhibitory effect on subsequent LCFA metabolism. First, the relative contribution of oxidation and esterification to LCFA uptake is not altered in the presence of SSO. Second, isoproterenol-mediated channeling of LCFAs into oxidative pathways is not affected by sulfo-N-succinimidyl palmitate (SSP). As an example of its application, we used SSP to study the role of FAT/CD36 in contraction- and insulin-stimulated LCFA uptake by cardiac myocytes, showing that this transporter is a primary site of regulation of cellular LCFA utilization.