RESUMO
The ability to feed on a sugar-containing diet depends on a gene regulatory network controlled by the intracellular sugar sensor Mondo/ChREBP-Mlx, which remains insufficiently characterized. Here, we present a genome-wide temporal clustering of sugar-responsive gene expression in Drosophila larvae. We identify gene expression programs responding to sugar feeding, including downregulation of ribosome biogenesis genes, known targets of Myc. Clockwork orange (CWO), a component of the circadian clock, is found to be a mediator of this repressive response and to be necessary for survival on a high-sugar diet. CWO expression is directly activated by Mondo-Mlx, and it counteracts Myc through repression of its gene expression and through binding to overlapping genomic regions. CWO mouse ortholog BHLHE41 has a conserved role in repressing ribosome biogenesis genes in primary hepatocytes. Collectively, our data uncover a cross-talk between conserved gene regulatory circuits balancing the activities of anabolic pathways to maintain homeostasis during sugar feeding.
Assuntos
Proteínas de Drosophila , Proteínas Repressoras , Ribossomos , Açúcares , Animais , Camundongos , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Ribossomos/metabolismo , Açúcares/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Repressoras/metabolismoRESUMO
BACKGROUND: Successful recovery from acute lung injury requires inhibition of neutrophil influx and clearance of apoptotic neutrophils. However, the mechanisms underlying recovery remain unclear. We investigated the ameliorative effects of vascular endothelial growth factor (VEGF)-C/VEGF receptor 3 (VEGFR-3) signalling in macrophages in lipopolysaccharide (LPS)-induced lung injury. METHODS: LPS was intranasally injected into wild-type and transgenic mice. Gain and loss of VEGF-C/VEGFR-3 signalling function experiments employed adenovirus-mediated intranasal delivery of VEGF-C (Ad-VEGF-C vector) and soluble VEGFR-3 (sVEGFR-3) or anti-VEGFR-3 blocking antibodies and mice with a deletion of VEGFR-3 in myeloid cells. RESULTS: The early phase of lung injury was significantly alleviated by the overexpression of VEGF-C with increased levels of bronchoalveolar lavage (BAL) fluid interleukin-10 (IL-10), but worsened in the later phase by VEGFR-3 inhibition upon administration of Ad-sVEGFR-3 vector. Injection of anti-VEGFR-3 antibodies to mice in the resolution phase inhibited recovery from lung injury. The VEGFR-3-deleted mice had a shorter survival time than littermates and more severe lung injury in the resolution phase. Alveolar macrophages in the resolution phase digested most of the extrinsic apoptotic neutrophils and VEGF-C/VEGFR-3 signalling increased efferocytosis via upregulation of integrin αv in the macrophages. We also found that incubation with BAL fluid from acute respiratory distress syndrome (ARDS) patients, but not from controls, decreased VEGFR-3 expression and the efficiency of IL-10 expression and efferocytosis in human monocyte-derived macrophages. CONCLUSIONS: VEGF-C/VEGFR-3 signalling in macrophages ameliorates experimental lung injury. This mechanism may also provide an explanation for ARDS resolution.
Assuntos
Lesão Pulmonar Aguda , Síndrome do Desconforto Respiratório , Lesão Pulmonar Aguda/metabolismo , Animais , Humanos , Interleucina-10/efeitos adversos , Interleucina-10/metabolismo , Lipopolissacarídeos , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Nutrient-dependent gene regulation critically contributes to homeostatic control of animal physiology in changing nutrient landscape. In Drosophila, dietary sugars activate transcription factors (TFs), such as Mondo-Mlx, Sugarbabe and Cabut, which control metabolic gene expression to mediate physiological adaptation to high sugar diet. TFs that correspondingly control sugar responsive metabolic genes under conditions of low dietary sugar remain, however, poorly understood. Here we identify a role for Drosophila GATA TF Grain in metabolic gene regulation under both low and high sugar conditions. De novo motif prediction uncovered a significant over-representation of GATA-like motifs on the promoters of sugar-activated genes in Drosophila larvae, which are regulated by Grain, the fly ortholog of GATA1/2/3 subfamily. grain expression is activated by sugar in Mondo-Mlx-dependent manner and it contributes to sugar-responsive gene expression in the fat body. On the other hand, grain displays strong constitutive expression in the anterior midgut, where it drives lipogenic gene expression also under low sugar conditions. Consistently with these differential tissue-specific roles, Grain deficient larvae display delayed development on high sugar diet, while showing deregulated central carbon and lipid metabolism primarily on low sugar diet. Collectively, our study provides evidence for the role of a metazoan GATA transcription factor in nutrient-responsive metabolic gene regulation in vivo.
Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Fatores de Transcrição GATA/genética , Animais , Regulação da Expressão Gênica/genética , Larva/genética , Açúcares/metabolismo , Ativação Transcricional/genéticaRESUMO
As ancient bacterial endosymbionts of eukaryotic cells, mitochondria have retained their own circular DNA as well as protein translation system including mitochondrial ribosomes (mitoribosomes). In recent years, methodological advancements in cryoelectron microscopy and mass spectrometry have revealed the extent of the evolutionary divergence of mitoribosomes from their bacterial ancestors and their adaptation to the synthesis of 13 mitochondrial DNA encoded oxidative phosphorylation complex subunits. In addition to the structural data, the first assembly pathway maps of mitoribosomes have started to emerge and concomitantly also the assembly factors involved in this process to achieve fully translational competent particles. These transiently associated factors assist in the intricate assembly process of mitoribosomes by enhancing protein incorporation, ribosomal RNA folding and modification, and by blocking premature or non-native protein binding, for example. This review focuses on summarizing the current understanding of the known mammalian mitoribosome assembly factors and discussing their possible roles in the assembly of small or large mitoribosomal subunits.
Assuntos
Genoma Mitocondrial , Mamíferos/genética , Mamíferos/fisiologia , Ribossomos Mitocondriais/fisiologia , AnimaisRESUMO
BACKGROUND: Heart failure, which is a major global health problem, is often preceded by pathological cardiac hypertrophy. The expansion of the cardiac vasculature, to maintain adequate supply of oxygen and nutrients, is a key determinant of whether the heart grows in a physiological compensated manner or a pathological decompensated manner. Bidirectional endothelial cell (EC)-cardiomyocyte (CMC) cross talk via cardiokine and angiocrine signaling plays an essential role in the regulation of cardiac growth and homeostasis. Currently, the mechanisms involved in the EC-CMC interaction are not fully understood, and very little is known about the EC-derived signals involved. Understanding how an excess of angiogenesis induces cardiac hypertrophy and how ECs regulate CMC homeostasis could provide novel therapeutic targets for heart failure. METHODS: Genetic mouse models were used to delete vascular endothelial growth factor (VEGF) receptors, adeno-associated viral vectors to transduce the myocardium, and pharmacological inhibitors to block VEGF and ErbB signaling in vivo. Cell culture experiments were used for mechanistic studies, and quantitative polymerase chain reaction, microarrays, ELISA, and immunohistochemistry were used to analyze the cardiac phenotypes. RESULTS: Both EC deletion of VEGF receptor (VEGFR)-1 and adeno-associated viral vector-mediated delivery of the VEGFR1-specific ligands VEGF-B or placental growth factor into the myocardium increased the coronary vasculature and induced CMC hypertrophy in adult mice. The resulting cardiac hypertrophy was physiological, as indicated by preserved cardiac function and exercise capacity and lack of pathological gene activation. These changes were mediated by increased VEGF signaling via endothelial VEGFR2, because the effects of VEGF-B and placental growth factor on both angiogenesis and CMC growth were fully inhibited by treatment with antibodies blocking VEGFR2 or by endothelial deletion of VEGFR2. To identify activated pathways downstream of VEGFR2, whole-genome transcriptomics and secretome analyses were performed, and the Notch and ErbB pathways were shown to be involved in transducing signals for EC-CMC cross talk in response to angiogenesis. Pharmacological or genetic blocking of ErbB signaling also inhibited part of the VEGF-B-induced effects in the heart. CONCLUSIONS: This study reveals that cross talk between the EC VEGFR2 and CMC ErbB signaling pathways coordinates CMC hypertrophy with angiogenesis, contributing to physiological cardiac growth.
Assuntos
Cardiomegalia/metabolismo , Células Endoteliais/metabolismo , Miócitos Cardíacos/metabolismo , Neovascularização Fisiológica , Comunicação Parácrina , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/patologia , Receptores ErbB/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/patologia , Receptor Cross-Talk , Receptores Notch/metabolismo , Fator B de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Lymphangiogenesis is supported by 2 homologous VEGFR3 ligands, VEGFC and VEGFD. VEGFC is required for lymphatic development, while VEGFD is not. VEGFC and VEGFD are proteolytically cleaved after cell secretion in vitro, and recent studies have implicated the protease a disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS3) and the secreted factor collagen and calcium binding EGF domains 1 (CCBE1) in this process. It is not well understood how ligand proteolysis is controlled at the molecular level or how this process regulates lymphangiogenesis, because these complex molecular interactions have been difficult to follow ex vivo and test in vivo. Here, we have developed and used biochemical and cellular tools to demonstrate that an ADAMTS3-CCBE1 complex can form independently of VEGFR3 and is required to convert VEGFC, but not VEGFD, into an active ligand. Consistent with these ex vivo findings, mouse genetic studies revealed that ADAMTS3 is required for lymphatic development in a manner that is identical to the requirement of VEGFC and CCBE1 for lymphatic development. Moreover, CCBE1 was required for in vivo lymphangiogenesis stimulated by VEGFC but not VEGFD. Together, these studies reveal that lymphangiogenesis is regulated by two distinct proteolytic mechanisms of ligand activation: one in which VEGFC activation by ADAMTS3 and CCBE1 spatially and temporally patterns developing lymphatics, and one in which VEGFD activation by a distinct proteolytic mechanism may be stimulated during inflammatory lymphatic growth.
Assuntos
Linfangiogênese/fisiologia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Fator D de Crescimento do Endotélio Vascular/metabolismo , Proteínas ADAMTS/deficiência , Proteínas ADAMTS/genética , Proteínas ADAMTS/metabolismo , Animais , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proliferação de Células , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células HEK293 , Humanos , Ligantes , Linfangiogênese/genética , Vasos Linfáticos/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Peptídeo Hidrolases/metabolismo , Pró-Colágeno N-Endopeptidase/genética , Pró-Colágeno N-Endopeptidase/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Fator C de Crescimento do Endotélio Vascular/deficiência , Fator C de Crescimento do Endotélio Vascular/genética , Fator D de Crescimento do Endotélio Vascular/deficiência , Fator D de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Impaired angiogenesis has been implicated in adipose tissue dysfunction and the development of obesity and associated metabolic disorders. Here, we report the unexpected finding that vascular endothelial growth factor B (VEGFB) gene transduction into mice inhibits obesity-associated inflammation and improves metabolic health without changes in body weight or ectopic lipid deposition. Mechanistically, the binding of VEGFB to VEGF receptor 1 (VEGFR1, also known as Flt1) activated the VEGF/VEGFR2 pathway and increased capillary density, tissue perfusion, and insulin supply, signaling, and function in adipose tissue. Furthermore, endothelial Flt1 gene deletion enhanced the effect of VEGFB, activating the thermogenic program in subcutaneous adipose tissue, which increased the basal metabolic rate, thus preventing diet-induced obesity and related metabolic complications. In obese and insulin-resistant mice, Vegfb gene transfer, together with endothelial Flt1 gene deletion, induced weight loss and mitigated the metabolic complications, demonstrating the therapeutic potential of the VEGFB/VEGFR1 pathway.
Assuntos
Tecido Adiposo/irrigação sanguínea , Obesidade/metabolismo , Fator B de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica , Obesidade/complicações , Obesidade/patologiaRESUMO
The mammalian circulatory system comprises both the cardiovascular system and the lymphatic system. In contrast to the blood vascular circulation, the lymphatic system forms a unidirectional transit pathway from the extracellular space to the venous system. It actively regulates tissue fluid homeostasis, absorption of gastrointestinal lipids, and trafficking of antigen-presenting cells and lymphocytes to lymphoid organs and on to the systemic circulation. The cardinal manifestation of lymphatic malfunction is lymphedema. Recent research has implicated the lymphatic system in the pathogenesis of cardiovascular diseases including obesity and metabolic disease, dyslipidemia, inflammation, atherosclerosis, hypertension, and myocardial infarction. Here, we review the most recent advances in the field of lymphatic vascular biology, with a focus on cardiovascular disease.
Assuntos
Doenças Cardiovasculares/fisiopatologia , Sistema Cardiovascular/fisiopatologia , Linfangiogênese , Sistema Linfático/fisiopatologia , Linfedema/fisiopatologia , Animais , Doenças Cardiovasculares/imunologia , Doenças Cardiovasculares/metabolismo , Sistema Cardiovascular/imunologia , Sistema Cardiovascular/metabolismo , Humanos , Sistema Linfático/imunologia , Sistema Linfático/metabolismo , Linfedema/imunologia , Linfedema/metabolismo , Transdução de SinaisRESUMO
Vascular endothelial growth factor C (VEGF-C) binding to its tyrosine kinase receptor VEGFR-3 drives lymphatic vessel growth during development and in pathological processes. Although the VEGF-C/VEGFR-3 pathway provides a target for treatment of cancer and lymphedema, the physiological functions of VEGF-C in adult vasculature are unknown. We show here that VEGF-C is necessary for perinatal lymphangiogenesis, but required for adult lymphatic vessel maintenance only in the intestine. Following Vegfc gene deletion in adult mice, the intestinal lymphatic vessels, including the lacteal vessels, underwent gradual atrophy, which was aggravated when also Vegfd was deleted. VEGF-C was expressed by a subset of smooth muscle cells adjacent to the lacteals in the villus and in the intestinal wall. The Vegfc-deleted mice showed defective lipid absorption and increased fecal excretion of dietary cholesterol and fatty acids. When fed a high-fat diet, the Vegfc-deficient mice were resistant to obesity and had improved glucose metabolism. Our findings indicate that the lymphangiogenic growth factors provide trophic and dynamic regulation of the intestinal lymphatic vasculature, which could be especially important in the dietary regulation of adiposity and cholesterol metabolism.
Assuntos
Intestinos/fisiologia , Metabolismo dos Lipídeos , Vasos Linfáticos/fisiologia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Animais , Deleção de Genes , Camundongos , Camundongos Knockout , Fator C de Crescimento do Endotélio Vascular/genéticaRESUMO
OBJECTIVE: Elevated serum levels of the lymphangiogenic factors VEGF-C and -D have been observed in obese individuals but their relevance for the metabolic syndrome has remained unknown. METHODS: K14-VEGFR-3-Ig (sR3) mice that constitutively express soluble-VEGFR-3-Ig in the skin, scavenging VEGF-C and -D, and wildtype (WT) mice were fed either chow or high-fat diet for 20 weeks. To assess the effect of VEGFR-3 blockage on adipose tissue growth and insulin sensitivity, we evaluated weight gain, adipocyte size and hepatic lipid accumulation. These results were complemented with insulin tolerance tests, FACS analysis of adipose tissue macrophages, in vitro 3T3-L1 differentiation assays and in vivo blocking antibody treatment experiments. RESULTS: We show here that sR3 mice are protected from obesity-induced insulin resistance and hepatic lipid accumulation. This protection is associated with enhanced subcutaneous adipose tissue hyperplasia and an increased number of alternatively-activated (M2) macrophages in adipose tissue. We also show that VEGF-C and -D are chemotactic for murine macrophages and that this effect is mediated by VEGFR-3, which is upregulated on M1 polarized macrophages. Systemic antibody blockage of VEGFR-3 in db/db mice reduces adipose tissue macrophage infiltration and hepatic lipid accumulation, and improves insulin sensitivity. CONCLUSIONS: These results reveal an unanticipated role of the lymphangiogenic factors VEGF-C and -D in the mediation of metabolic syndrome-associated adipose tissue inflammation. Blockage of these lymphangiogenic factors might constitute a new therapeutic strategy for the prevention of obesity-associated insulin resistance.
RESUMO
OBJECTIVE: Lymphatic vessels collect extravasated fluid and proteins from tissues to blood circulation as well as play an essential role in lipid metabolism by taking up intestinal chylomicrons. Previous studies have shown that impairment of lymphatic vessel function causes lymphedema and fat accumulation, but clear connections between arterial pathologies and lymphatic vessels have not been described. APPROACH AND RESULTS: Two transgenic mouse strains with lymphatic insufficiency (soluble vascular endothelial growth factor 3 [sVEGFR3] and Chy) were crossed with atherosclerotic mice deficient of low-density lipoprotein receptor and apolipoprotein B48 (LDLR(-/-)/ApoB(100/100)) to study the effects of insufficient lymphatic vessel transport on lipoprotein metabolism and atherosclerosis. Both sVEGFR3×LDLR(-/-)/ApoB(100/100) mice and Chy×LDLR(-/-)/ApoB(100/100) mice had higher plasma cholesterol levels compared with LDLR(-/-)/ApoB(100/100) control mice during both normal chow diet (16.3 and 13.7 versus 8.2 mmol/L, respectively) and Western-type high-fat diet (eg, after 2 weeks of fat diet, 45.9 and 42.6 versus 30.2 mmol/L, respectively). Cholesterol and triglyceride levels in very-low-density lipoprotein and low-density lipoprotein fractions were increased. Atherosclerotic lesions in young and intermediate cohorts of sVEGFR3×LDLR(-/-)/ApoB(100/100) mice progressed faster than in control mice (eg, intermediate cohort mice at 6 weeks, 18.3% versus 7.7% of the whole aorta, respectively). In addition, lesions in sVEGFR3×LDLR(-/-)/ApoB(100/100) mice and Chy×LDLR(-/-)/ApoB(100/100) mice had much less lymphatic vessels than lesions in control mice (0.33% and 1.07% versus 7.45% of podoplanin-positive vessels, respectively). CONCLUSIONS: We show a novel finding linking impaired lymphatic vessels to lipoprotein metabolism, increased plasma cholesterol levels, and enhanced atherogenesis.
Assuntos
Aterosclerose/etiologia , Hipercolesterolemia/complicações , Lipoproteínas/metabolismo , Vasos Linfáticos/fisiopatologia , Animais , Apolipoproteínas B/fisiologia , Colesterol/metabolismo , Humanos , Lipídeos/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de LDL/fisiologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Angiogenic growth factors have recently been linked to tissue metabolism. We have used genetic gain- and loss-of function models to elucidate the effects and mechanisms of action of vascular endothelial growth factor-B (VEGF-B) in the heart. A cardiomyocyte-specific VEGF-B transgene induced an expanded coronary arterial tree and reprogramming of cardiomyocyte metabolism. This was associated with protection against myocardial infarction and preservation of mitochondrial complex I function upon ischemia-reperfusion. VEGF-B increased VEGF signals via VEGF receptor-2 to activate Erk1/2, which resulted in vascular growth. Akt and mTORC1 pathways were upregulated and AMPK downregulated, readjusting cardiomyocyte metabolic pathways to favor glucose oxidation and macromolecular biosynthesis. However, contrasting with a previous theory, there was no difference in fatty acid uptake by the heart between the VEGF-B transgenic, gene-targeted or wildtype rats. Importantly, we also show that VEGF-B expression is reduced in human heart disease. Our data indicate that VEGF-B could be used to increase the coronary vasculature and to reprogram myocardial metabolism to improve cardiac function in ischemic heart disease.
Assuntos
Isquemia Miocárdica/prevenção & controle , Miocárdio/metabolismo , Fator B de Crescimento do Endotélio Vascular/metabolismo , Adenoviridae/genética , Animais , Vetores Genéticos/metabolismo , Coração/diagnóstico por imagem , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Infarto do Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Ratos Wistar , Transdução de Sinais , Tomografia Computadorizada por Raios X , Regulação para Cima , Fator B de Crescimento do Endotélio Vascular/deficiência , Fator B de Crescimento do Endotélio Vascular/genéticaRESUMO
OBJECTIVE: Angiopoietin-like 3 (Angptl3) is a regulator of lipoprotein metabolism at least by inhibiting lipoprotein lipase activity. Loss-of-function mutations in ANGPTL3 cause familial combined hypolipidemia through an unknown mechanism. APPROACH AND RESULTS: We compared lipolytic activities, lipoprotein composition, and other lipid-related enzyme/lipid transfer proteins in carriers of the S17X loss-of-function mutation in ANGPTL3 and in age- and sex-matched noncarrier controls. Gel filtration analysis revealed a severely disturbed lipoprotein profile and a reduction in size and triglyceride content of very low density lipoprotein in homozygotes as compared with heterozygotes and noncarriers. S17X homozygotes had significantly higher lipoprotein lipase activity and mass in postheparin plasma, whereas heterozygotes showed no difference in these parameters when compared with noncarriers. No changes in hepatic lipase, endothelial lipase, paraoxonase 1, phospholipid transfer protein, and cholesterol ester transfer protein activities were associated with the S17X mutation. Plasma free fatty acid, insulin, glucose, and homeostatic model assessment of insulin resistance were significantly lower in homozygous subjects compared with heterozygotes and noncarriers subjects. CONCLUSIONS: These results indicate that, although partial Angptl3 deficiency did not affect the activities of lipolytic enzymes, the complete absence of Angptl3 results in an increased lipoprotein lipase activity and mass and low circulating free fatty acid levels. This latter effect is probably because of decreased mobilization of free fatty acid from fat stores in human adipose tissue and may result in reduced hepatic very low density lipoprotein synthesis and secretion via attenuated hepatic free fatty acid supply. Altogether, Angptl3 may affect insulin sensitivity and play a role in modulating both lipid and glucose metabolism.
Assuntos
Angiopoietinas/deficiência , Ácidos Graxos não Esterificados/sangue , Hipobetalipoproteinemias/enzimologia , Resistência à Insulina , Lipase Lipoproteica/sangue , Adulto , Idoso , Análise de Variância , Proteína 3 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/genética , Biomarcadores/sangue , Glicemia/análise , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Regulação para Baixo , Feminino , Heterozigoto , Homozigoto , Humanos , Hipobetalipoproteinemias/sangue , Hipobetalipoproteinemias/genética , Hipobetalipoproteinemias/fisiopatologia , Insulina/sangue , Itália , Modelos Lineares , Lipase/sangue , Lipoproteínas LDL/sangue , Masculino , Pessoa de Meia-Idade , Mutação , Triglicerídeos/sangue , Regulação para CimaRESUMO
Peroxisome proliferator-activated receptor (PPAR) delta is an important regulator of fatty acid (FA) metabolism. Angiopoietin-like 4 (Angptl4), a multifunctional protein, is one of the major targets of PPAR delta in skeletal muscle cells. Here we investigated the regulation of Angptl4 and its role in mediating PPAR delta functions using human, rat and mouse myotubes. Expression of Angptl4 was upregulated during myotubes differentiation and by oleic acid, insulin and PPAR delta agonist GW501516. Treatment with GW501516 or Angptl4 overexpression inhibited both lipoprotein lipase (LPL) activity and LPL-dependent uptake of FAs whereas uptake of BSA-bound FAs was not affected by either treatment. Activation of retinoic X receptor (RXR), PPAR delta functional partner, using bexarotene upregulated Angptl4 expression and inhibited LPL activity in a PPAR delta dependent fashion. Silencing of Angptl4 blocked the effect of GW501516 and bexarotene on LPL activity. Treatment with GW501516 but not Angptl4 overexpression significantly increased palmitate oxidation. Furthermore, Angptl4 overexpression did not affect the capacity of GW501516 to increase palmitate oxidation. Basal and insulin stimulated glucose uptake, glycogen synthesis and glucose oxidation were not significantly modulated by Angptl4 overexpression. Our findings suggest that FAs-PPARdelta/RXR-Angptl4 axis controls the LPL-dependent uptake of FAs in myotubes, whereas the effect of PPAR delta activation on beta-oxidation is independent of Angptl4.
Assuntos
Angiopoietinas/metabolismo , Ácidos Graxos/metabolismo , Lipase Lipoproteica/metabolismo , Fibras Musculares Esqueléticas/metabolismo , PPAR delta/metabolismo , Proteína 4 Semelhante a Angiopoietina , Angiopoietinas/genética , Animais , Bexaroteno , Western Blotting , Linhagem Celular , Células Cultivadas , Ácidos Graxos/farmacocinética , Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Oxirredução , PPAR delta/agonistas , PPAR delta/genética , Palmitatos/metabolismo , Interferência de RNA , Ratos , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tetra-Hidronaftalenos/farmacologia , Tiazóis/farmacologiaRESUMO
Oxysterol-binding protein (OSBP) homologues, ORPs, are implicated in lipid homeostatic control, vesicle transport, and cell signaling. We analyzed here the quantity of ORP mRNAs in human subcutaneous (s.c.) and visceral adipose depots, as well as in the Simpson-Golabi-Behmel syndrome (SGBS) adipocyte cell model. All of the ORP mRNAs were present in the s.c and visceral adipose tissues, and the two depots shared an almost identical ORP mRNA expression pattern. SGBS adipocytes displayed a similar pattern, suggesting that the adipose tissue ORP expression pattern mainly derives from adipocytes. During SGBS cell adipogenic differentiation, ORP2, ORP3, ORP4, ORP7, and ORP8 mRNAs were down-regulated, while ORP11 was induced. To assess the impacts of ORPs on adipocyte differentiation, ORP3 and ORP8, proteins down-regulated during adipogenesis, were overexpressed in differentiating SGBS adipocytes, while ORP11, a protein induced during adipogenesis, was silenced. ORP8 overexpression resulted in reduced expression of the aP2 mRNA, while down-regulation of adiponectin and aP2 was observed in ORP11 silenced cells. Furthermore, ORP8 overexpression or silencing of ORP11 markedly decreased cellular triglyceride storage. These data identify the patterns of ORP expression in human adipose depots and SGBS adipocytes, and provide the first evidence for a functional impact of ORPs on the adipocyte phenotype.
Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Receptores de Esteroides/metabolismo , Adiponectina/metabolismo , Adulto , Western Blotting , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Biologia Computacional , Proteínas de Ligação a Ácido Graxo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Receptores de Esteroides/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Triglicerídeos/metabolismoRESUMO
CONTEXT: Familial combined hypolipidemia causes a global reduction of plasma lipoproteins. Its clinical correlates and metabolic implications have not been well defined. OBJECTIVE: The objective of the study was to investigate the genetic, clinical, and metabolic characteristics of a cohort of subjects with familial combined hypolipidemia. DESIGN: The design of the study included candidate gene screening and the comparison of the clinical and metabolic characteristics between carrier and noncarrier individuals. SETTING: The study was conducted in a general community. SUBJECTS: Participants in the study included individuals belonging to nine families with familial combined hypolipidemia identified in a small town (Campodimele) as well as from other 352 subjects living in the same community. MAIN OUTCOMES MEASURES: Serum concentrations of lipoproteins, Angiopoietin-like 3 (Angptl3) proteins, and noncholesterol sterols were measured. RESULTS: The ANGPTL3 S17X mutation was found in all probands, 20 affected family members, and 32 individuals of the community. Two additional frame shift mutations, FsE96del and FsS122, were also identified in two hypocholesterolemic individuals. Homozygotes for the ANGPTL3 S17X mutation had no circulating Angptl3 and a marked reduction of all plasma lipids (P < 0.001). Heterozygotes had 42% reduction in Angptl3 level compared with noncarriers (P < 0.0001) but a significant reduction of only total cholesterol and high-density lipoprotein cholesterol. No differences were observed in the plasma noncholesterol sterols between carriers and noncarriers. No association between familial combined hypolipidemia and the risk of hepatic or cardiovascular diseases were detected. CONCLUSIONS: Familial combined hypolipidemia segregates as a recessive trait so that apolipoprotein B- and apolipoprotein A-I-containing lipoproteins are comprehensively affected only by the total deficiency of Angptl3. Familial combined hypolipidemia does not perturb whole-body cholesterol homeostasis and is not associated with adverse clinical sequelae.
Assuntos
Angiopoietinas/genética , Hipolipoproteinemias/genética , Mutação , Adulto , Idoso , Proteína 3 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Angiopoietinas/sangue , Apolipoproteína A-I/sangue , Apolipoproteína A-I/metabolismo , Apolipoproteínas B/sangue , Apolipoproteínas B/metabolismo , Estudos de Casos e Controles , Colesterol/sangue , Estudos de Coortes , Análise Mutacional de DNA , Família , Feminino , Genótipo , Humanos , Hipolipoproteinemias/sangue , Hipolipoproteinemias/diagnóstico , Hipolipoproteinemias/epidemiologia , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Mutação/fisiologia , Linhagem , FenótipoRESUMO
Cholesteryl ester transfer protein (CETP) and apolipoprotein E (apoE) are secreted by macrophages. Apolipoprotein A-I (apoA-I) is a potent inducer of apoE secretion from lipid-loaded macrophages, but its effect on CETP is not known. We aimed to identify the signaling pathways involved in apoA-I and HDL-mediated regulation of CETP and apoE secretion from lipid-loaded macrophages. THP-1 macrophages were loaded with lipids by incubation with human copper-oxidized LDL. The cells were subsequently exposed to human purified apoA-I or HDL(3) with/without inhibitors of NF-κB (TPCK) or PKA (H89). CETP and apoE in the cultured cells and media were quantified by real-time PCR and Western blot. Results showed that in lipid-loaded macrophages: (i) CETP and apoE gene expression and secretion were increased in the presence of apoA-I, and further increased by inhibition of NF-kB with TPCK; (ii) CETP and apoE gene expression and secretion were reduced by the inhibition of PKA with H89; (iii) PKA-gamma subunit was activated by oxidized LDL and moreover by apoA-I. We also showed that: (i) siRNA-mediated CETP gene silencing diminished apoE secretion from both non-loaded and lipid-loaded macrophages; (ii) addition of apoA-I partially restored apoE secretion from lipid-loaded macrophages with the silenced CETP gene. In conclusion, our data suggest a new mechanism by which apoA-I stimulates CETP secretion, in addition to apoE, from lipid loaded macrophages, a process involving NF-κB inhibition and/or PKA pathway activation.
Assuntos
Apolipoproteína A-I/metabolismo , Apolipoproteínas E/metabolismo , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , HDL-Colesterol/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Macrófagos/metabolismo , NF-kappa B/metabolismo , Apolipoproteína A-I/farmacologia , Proteínas de Transferência de Ésteres de Colesterol/genética , HDL-Colesterol/farmacologia , Expressão Gênica , Inativação Gênica , Humanos , Metabolismo dos Lipídeos , Macrófagos/efeitos dos fármacos , RNA Interferente Pequeno/genética , Transdução de SinaisRESUMO
Animal studies have suggested that angiopoietin-like 4 (Angptl4) regulates adiposity through central and peripheral mechanisms. The aim of this study was to investigate whether serum concentration and adipose tissue expression of Angptl4 are associated with obesity-related parameters in humans. Altogether, 75 dizygotic (DZ) and 46 monozygotic (MZ) twin pairs were studied, from the FinnTwin12 and FinnTwin16 cohorts. Among the MZ pairs, 21 were discordant for body mass index (BMI) (intra-pair BMI-difference >2.5 kg/m², age 23-33 years). Serum Angptl4 (s-Angptl4) levels were measured by ELISA, and adipose tissue gene expression was analyzed by genome-wide transcript profiling. In MZ twin pairs discordant for BMI, s-Angptl4 and adipose tissue ANGPTL4 mRNA (at-ANGPTL4) levels were significantly decreased (P = 0.04 and P = 0.03, respectively) in obese twins as compared with their nonobese cotwins. In all twins, intra-pair differences in s-Angptl4 levels were inversely correlated with intra-pair differences in BMI (r = -0.27, P = 0.003). In individual MZ twins, at-ANGPTL4 expression was inversely correlated with BMI (r = -0.44, P = 0.001) and positively correlated with at-LIPE (r = 0.24, P = 0.01) and at-ABHD5 (r = 0.41, P = 0.005) expression. Our results demonstrated that variation in Angptl4 concentration was only modestly accounted for by genetic factors and suggest a role for Angptl4 in acquired obesity in humans.
Assuntos
Tecido Adiposo/metabolismo , Angiopoietinas/metabolismo , Proteínas Sanguíneas/metabolismo , Obesidade , Gêmeos Dizigóticos/metabolismo , Gêmeos Monozigóticos/metabolismo , Proteína 4 Semelhante a Angiopoietina , Angiopoietinas/genética , Biópsia , Glicemia/análise , Proteínas Sanguíneas/genética , Composição Corporal , Índice de Massa Corporal , Feminino , Finlândia , Expressão Gênica , Humanos , Estudos Longitudinais , Masculino , Obesidade/sangue , Obesidade/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/análise , Gêmeos Dizigóticos/genética , Gêmeos Monozigóticos/genéticaRESUMO
OBJECTIVE: To investigate the relationship between angiopoietin-like protein 4 (Angptl4) levels, coronary heart disease (CHD) biomarkers, and ANGPTL4 variants. METHODS AND RESULTS: Plasma Angptl4 was quantified in 666 subjects of the Northwick Park Heart Study II using a validated ELISA. Seven ANGPTL4 single-nucleotide polymorphisms were genotyped, and CHD biomarkers were assessed in the whole cohort (N=2775). Weighted mean±SD plasma Angptl4 levels were 10.0±11.0 ng/mL. Plasma Angptl4 concentration correlated positively with age (r=0.15, P<0.001) and body fat mass (r=0.19, P=0.003) but negatively with plasma high-density lipoprotein cholesterol (r=-0.13, P=0.01). No correlation with triglycerides (TGs) was observed. T266M was independently associated with plasma Angptl4 levels (P<0.001) but was not associated with TGs or CHD risk in the meta-analysis of 5 studies (4061 cases/15 395 controls). E40K showed no independent association with plasma Angptl4 levels. In human embryonic kidney 293 and human hepatoma 7 cells compared with wild type, E40K and T266M showed significantly altered synthesis and secretion, respectively. CONCLUSIONS: Circulating Angptl4 levels may not influence TG levels or CHD risk for the following reasons: (1) Angptl4 levels were not correlated with TGs; (2) T266M, although associated with Angptl4 levels, showed no association with plasma TGs; and (3) TG-lowering E40K did not influence Angptl4 levels. These results provide new insights into the role of Angptl4 in TG metabolism.
Assuntos
Angiopoietinas/genética , Doença da Artéria Coronariana/genética , Proteína 4 Semelhante a Angiopoietina , Angiopoietinas/sangue , Biomarcadores/sangue , Linhagem Celular , Doença da Artéria Coronariana/sangue , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Fatores de RiscoRESUMO
BACKGROUND: phospholipid transfer protein (PLTP) plays important roles in lipoprotein metabolism and atherosclerosis and is expressed by macrophages and macrophage foam cells (MFCs). The aim of the present study was to determine whether the major protein from HDL, apoA-I, affects PLTP derived from MFCs. RESULTS: as cell model we used human THP-1 monocytes incubated with acetylated LDL, to generate MFC. The addition of apoA-I to the cell media increased apoE secretion from the cells, in a concentration dependent fashion, without affecting cellular apoE levels. In contrast, apoA-I had no effect on PLTP synthesis and secretion, but strongly induced the PLTP activity in the media. ApoA-I also increased phospholipid transfer activity of PLTP isolated from human plasma. This effect was dependent on apoA-I concentration but independent on apoA-I lipidation status. ApoE, ApoA-II and apoA-IV, but not immunoglobulins or bovine serum albumin, also increased PLTP activity. We also report that apoA-I protects PLTP from heat inactivation. CONCLUSION: apoA-I enhances the phospholipid transfer activity of PLTP secreted from macrophage foam cells without affecting the PLTP mass.