RESUMEN
Enzymes are effective biological catalysts that accelerate almost all metabolic reactions in living organisms. Synthetic modulators of enzymes are useful tools for the study of enzymatic reactions and can provide starting points for the design of new drugs. Here, we report on the discovery of a class of biologically active compounds that covalently modifies lysine residues in human liver pyruvate kinase (PKL), leading to allosteric activation of the enzyme (EC50 =0.29â µM). Surprisingly, the allosteric activation control point resides on the lysine residue K282 present in the catalytic site of PKL. These findings were confirmed by structural data, MS/MS experiments, and molecular modelling studies. Altogether, our study provides a molecular basis for the activation mechanism and establishes a framework for further development of human liver pyruvate kinase covalent activators.
Asunto(s)
Lisina , Piruvato Quinasa , Humanos , Piruvato Quinasa/química , Piruvato Quinasa/metabolismo , Espectrometría de Masas en Tándem , Hígado , Dominio Catalítico , Regulación AlostéricaRESUMEN
BACKGROUND: The initiating step in atherogenesis is the electrostatic binding of LDL (low-density lipoprotein) to proteoglycan glycosaminoglycans in the arterial intima. However, although proteoglycans are widespread throughout the intima of most coronary artery segments, LDL is not evenly distributed, indicating that LDL retention is not merely dependent on the presence of proteoglycans. We aim to identify factors that promote the interaction between LDL and the vessel wall of human coronary arteries. METHODS: We developed an ex vivo model to investigate binding of labeled human LDL to human coronary artery sections without the interference of cellular processes. RESULTS: By staining consecutive sections of human coronary arteries, we found strong staining of sulfated glycosaminoglycans throughout the arterial intima, whereas endogenous LDL deposits were focally distributed. Ex vivo binding of LDL was uniform at all intimal areas with sulfated glycosaminoglycans. However, lowering the pH from 7.4 to 6.5 triggered a 35-fold increase in LDL binding. The pH-dependent binding was abolished by pretreating LDL with diethyl-pyrocarbonate, which blocks the protonation of histidine residues, or cyclohexanedione, which inhibits the positive charge of site B on LDL. Thus, both histidine protonation and site B are required for strong electrostatic LDL binding to the intima. CONCLUSIONS: This study identifies histidine protonation as an important component for electrostatic LDL binding to human coronary arteries. Our findings show that the local pH will have a profound impact on LDL's affinity for sulfated glycosaminoglycans, which may influence the retention and accumulation pattern of LDL in the arterial vasculature.
Asunto(s)
Vasos Coronarios , Lipoproteínas LDL , Vasos Coronarios/metabolismo , Glicosaminoglicanos/metabolismo , Histidina , Humanos , Concentración de Iones de Hidrógeno , Lipoproteínas LDL/metabolismo , Proteoglicanos/metabolismo , Electricidad EstáticaRESUMEN
Lipid droplet formation, which is driven by triglyceride synthesis, requires several droplet-associated proteins. We identified ARAP2 (an ADP-ribosylation factor 6 GTPase-activating protein) in the lipid droplet proteome of NIH-3T3 cells and showed that knockdown of ARAP2 resulted in decreased lipid droplet formation and triglyceride synthesis. We also showed that ARAP2 knockdown did not affect fatty acid uptake but reduced basal glucose uptake, total levels of the glucose transporter GLUT1, and GLUT1 levels in the plasma membrane and the lipid micro-domain fraction (a specialized plasma membrane domain enriched in sphingolipids). Microarray analysis showed that ARAP2 knockdown altered expression of genes involved in sphingolipid metabolism. Because sphingolipids are known to play a key role in cell signaling, we performed lipidomics to further investigate the relationship between ARAP2 and sphingolipids and potentially identify a link with glucose uptake. We found that ARAP2 knockdown increased glucosylceramide and lactosylceramide levels without affecting ceramide levels, and thus speculated that the rate-limiting enzyme in glycosphingolipid synthesis, namely glucosylceramide synthase (GCS), could be modified by ARAP2. In agreement with our hypothesis, we showed that the activity of GCS was increased by ARAP2 knockdown and reduced by ARAP2 overexpression. Furthermore, pharmacological inhibition of GCS resulted in increases in basal glucose uptake, total GLUT1 levels, triglyceride biosynthesis from glucose, and lipid droplet formation, indicating that the effects of GCS inhibition are the opposite to those resulting from ARAP2 knockdown. Taken together, our data suggest that ARAP2 promotes lipid droplet formation by modifying sphingolipid metabolism through GCS.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Glucosa/metabolismo , Metabolismo de los Lípidos , Esfingolípidos/metabolismo , Factor 6 de Ribosilación del ADP , Animales , Membrana Celular/metabolismo , Proteínas Activadoras de GTPasa/química , Técnicas de Silenciamiento del Gen , Glucosilceramidas/metabolismo , Gotas Lipídicas/metabolismo , Microdominios de Membrana/metabolismo , Ratones , Células 3T3 NIH , Dominios Homólogos a Pleckstrina , Dominios Proteicos , Proteoma/metabolismo , Proteómica , Triglicéridos/biosíntesisRESUMEN
Non-alcoholic fatty liver disease (NAFLD) is a prevalent pathological condition characterised by the accumulation of fat in the liver. Almost one-third of the global population is affected by NAFLD, making it a significant health concern. However, despite its prevalence, there is currently no approved drug specifically designed for the treatment of NAFLD. To address this critical gap, researchers have been investigating potential targets for NAFLD drug development. One promising candidate is the liver isoform of pyruvate kinase (PKL). In recent studies, Urolithin C, an allosteric inhibitor of PKL, has emerged as a potential lead compound for therapeutic intervention. Building upon this knowledge, our team has conducted a comprehensive structure-activity relationship of Urolithin C. In this work, we have employed a scaffold-hopping approach, modifying the urolithin structure by replacing the urolithin carbonyl with a sulfone moiety. Our structure-activity relationship analysis has identified the sulfone group as particularly favourable for potent PKL inhibition. Additionally, we have found that the presence of catechol moieties on the two aromatic rings further improves the inhibitory activity. The most promising inhibitor from this new series displayed nanomolar inhibition, boasting an IC50 value of 0.07 µM. This level of potency rivals that of urolithin D and significantly surpasses the effectiveness of urolithin C by an order of magnitude. To better understand the molecular interactions underlying this inhibition, we obtained the crystal structure of one of the inhibitors complexed with PKL. This structural insight served as a valuable reference point, aiding us in the design of inhibitors.
Asunto(s)
Taninos Hidrolizables , Enfermedad del Hígado Graso no Alcohólico , Piruvato Quinasa , Humanos , Hígado , Sulfonas/farmacologíaRESUMEN
The liver isoform of pyruvate kinase (PKL) has gained interest due to its potential capacity to regulate fatty acid synthesis involved in the progression of non-alcoholic fatty liver disease (NAFLD). Here we describe a novel series of PKL modulators that can either activate or inhibit the enzyme allosterically, from a cryptic site at the interface of two protomers in the tetrameric enzyme. Starting from urolithin D, we designed and synthesised 42 new compounds. The effect of these compounds on PKL enzymatic activity was assessed after incubation with cell lysates obtained from a liver cell line. Pronounced activation of PKL activity, up to 3.8-fold, was observed for several compounds at 10 µM, while other compounds were prominent PKL inhibitors reducing its activity to 81% at best. A structure-activity relationship identified linear-shaped sulfone-sulfonamides as activators and non-linear compounds as inhibitors. Crystal structures revealed the conformations of these modulators, which were used as a reference for designing new modulators.
Asunto(s)
Hígado , Piruvato Quinasa , Piruvato Quinasa/metabolismo , Hígado/metabolismo , Hepatocitos/metabolismo , Línea Celular , LipogénesisRESUMEN
The inhibition of liver pyruvate kinase could be beneficial to halt or reverse non-alcoholic fatty liver disease (NAFLD), a progressive accumulation of fat in the liver that can lead eventually to cirrhosis. Recently, urolithin C has been reported as a new scaffold for the development of allosteric inhibitors of liver pyruvate kinase (PKL). In this work, a comprehensive structure-activity analysis of urolithin C was carried out. More than 50 analogues were synthesized and tested regarding the chemical features responsible for the desired activity. These data could pave the way to the development of more potent and selective PKL allosteric inhibitors.
RESUMEN
REarranged during Transfection (RET) is a transmembrane receptor tyrosine kinase that is required for development of multiple human tissues, but which is also an important contributor to human cancers. RET activation through rearrangement or point mutations occurs in thyroid and lung cancers. Furthermore, activation of wild type RET is an increasingly recognized mechanism promoting tumor growth and dissemination of a much broader group of cancers. RET is therefore an attractive therapeutic target for small-molecule kinase inhibitors. Non-invasive control of RET signaling with light offers the promise of unveiling its complex spatiotemporal dynamics in vivo. In this work, photoswitchable DFG-out RET kinase inhibitors based on heterocycle-derived azobenzenes were developed, enabling photonic control of RET activity. Based on the binding mode of DFG-out kinase inhibitors and using RET kinase as the test model, we developed a photoswitchable inhibitor with a quinoline "head" constituting the azoheteroarene. This azo compound was further modified by three different strategies to increase the difference in biological activity between the E-isomer and the light enriched Z-isomer. Stilbene-based derivatives were used as model compounds to guide in the selection of substituents that could eventually be introduced to the corresponding azo compounds. The most promising quinoline-based compound showed more than a 15-fold difference in bioactivity between the two isomers in a biochemical assay. However, the same compound showed a decreased Z/E (IC50) ratio in the cellular assay, tentatively assigned to stability issues. The corresponding stilbene compound gave a Z/E (IC50) ratio well above 100, consistent with that measured in the biochemical assay. Ultimately, a 7-azaindole based photoswitchable DFG-out kinase inhibitor was shown to display more than a 10-fold difference in bioactivity between the two isomers, in both a biochemical and a cell-based assay, as well as excellent stability even under reducing conditions.
Asunto(s)
Antineoplásicos , Neoplasias Pulmonares , Estilbenos , Antineoplásicos/farmacología , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas c-ret , Proteínas Tirosina Quinasas ReceptorasRESUMEN
Liver pyruvate kinase (PKL) is a major regulator of metabolic flux and ATP production during liver cell glycolysis and is considered a potential drug target for the treatment of non-alcoholic fatty liver disease (NAFLD). In this study, we report the first ADP-competitive PKL inhibitors and identify several starting points for the further optimization of these inhibitors. Modeling and structural biology guided the optimization of a PKL-specific anthraquinone-based compound. A structure-activity relationship study of 47 novel synthetic derivatives revealed PKL inhibitors with half-maximal inhibitory concentration (IC50) values in the 200 nM range. Despite the difficulty involved in studying a binding site as exposed as the ADP site, these derivatives feature expanded structural diversity and chemical spaces that may be used to improve their inhibitory activities against PKL. The obtained results expand the knowledge of the structural requirements for interactions with the ADP-binding site of PKL.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Piruvato Quinasa , Adenosina Difosfato/farmacología , Antraquinonas/farmacología , Humanos , Hígado/metabolismo , Piruvato Quinasa/metabolismoRESUMEN
We report the synthesis and characterisation of a photoswitchable DFG-out kinase inhibitor. Photocontrol of the target kinase in both enzymatic and living cell assays is demonstrated.
Asunto(s)
Compuestos Azo/farmacología , Desarrollo de Medicamentos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Compuestos Azo/química , Células HEK293 , Humanos , Estructura Molecular , Inhibidores de Proteínas Quinasas/químicaRESUMEN
The Rho GTPase RAC1 is an important regulator of cytoskeletal dynamics, but the role of macrophage-specific RAC1 has not been explored during atherogenesis. We analyzed RAC1 expression in human carotid atherosclerotic plaques using immunofluorescence and found higher macrophage RAC1 expression in advanced plaques compared with intermediate human atherosclerotic plaques. We then produced mice with Rac1-deficient macrophages by breeding conditional floxed Rac1 mice (Rac1fl/fl) with mice expressing Cre from the macrophage-specific lysosome M promoter (LC). Atherosclerosis was studied in vivo by infecting Rac1fl/fl and Rac1fl/fl/LC mice with AdPCSK9 (adenoviral vector overexpressing proprotein convertase subtilisin/kexin type 9). Rac1fl/fl/LC macrophages secreted lower levels of IL-6 and TNF-α and exhibited reduced foam cell formation and lipid uptake. The deficiency of Rac1 in macrophages reduced the size of aortic atherosclerotic plaques in AdPCSK9-infected Rac1fl/fl/LC mice. Compare with controls, intima/media ratios, the size of necrotic cores, and numbers of CD68-positive macrophages in atherosclerotic plaques were reduced in Rac1-deficient mice. Moreover, we found that RAC1 interacts with actin-binding filamin A. Macrophages expressed increased RAC1 levels in advanced human atherosclerosis. Genetic inactivation of RAC1 impaired macrophage function and reduced atherosclerosis in mice, suggesting that drugs targeting RAC1 may be useful in the treatment of atherosclerosis.
Asunto(s)
Aterosclerosis/metabolismo , Macrófagos/metabolismo , Neuropéptidos/genética , Proteína de Unión al GTP rac1/genética , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Aterosclerosis/genética , Aterosclerosis/patología , Células Cultivadas , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Metabolismo de los Lípidos , Ratones , Ratones Endogámicos C57BL , Neuropéptidos/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Toll-like receptor 4 (TLR4) is activated by bacterial lipopolysaccharide (LPS), which drives the production of proinflammatory cytokines. Earlier studies have indicated that cholesterol- and glycosphingolipid-rich subregions of the plasma membrane (lipid domains) are important for TLR4-mediated signaling. We report that inhibition of glucosylceramide (GluCer) synthase, which resulted in decreased concentrations of the glycosphingolipid GluCer in lipid domains, reduced the LPS-induced inflammatory response in both mouse and human macrophages. Atomistic molecular dynamics simulations of the TLR4 dimer complex (with and without LPS in its MD-2 binding pockets) in membranes (in the presence and absence of GluCer) showed that: (1) LPS induced a tilted orientation of TLR4 and increased dimer integrity; (2) GluCer did not affect the integrity of the LPS/TLR4 dimer but reduced the LPS-induced tilt; and (3) GluCer increased electrostatic interactions between the membrane and the TLR4 extracellular domain, which could potentially modulate the tilt. We also showed that GCS inhibition reduced the interaction between TLR4 and the intracellular adaptor protein Mal. We conclude that the GluCer-induced effects on LPS/TLR4 orientation may influence the signaling capabilities of the LPS/TLR4 complex by affecting its interaction with downstream signaling proteins.
Asunto(s)
Glucosilceramidas/química , Glucosiltransferasas/química , Lipopolisacáridos/química , Macrófagos/inmunología , Simulación de Dinámica Molecular , Receptor Toll-Like 4/química , Animales , Sitios de Unión , Diferenciación Celular/efectos de los fármacos , Membrana Celular/química , Membrana Celular/inmunología , Membrana Celular/metabolismo , Expresión Génica , Glucosilceramidas/inmunología , Glucosilceramidas/metabolismo , Glucosiltransferasas/antagonistas & inhibidores , Glucosiltransferasas/genética , Glucosiltransferasas/inmunología , Células HEK293 , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Antígeno 96 de los Linfocitos/química , Antígeno 96 de los Linfocitos/genética , Antígeno 96 de los Linfocitos/inmunología , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito/química , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito/genética , Proteínas Proteolipídicas Asociadas a Mielina y Linfocito/inmunología , Cultivo Primario de Células , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunologíaRESUMEN
The aim was to clarify the role of vimentin, an intermediate filament protein abundantly expressed in activated macrophages and foam cells, in macrophages during atherogenesis. Global gene expression, lipid uptake, ROS, and inflammation were analyzed in bone-marrow derived macrophages from vimentin-deficient (Vim-/-) and wild-type (Vim+/+) mice. Atherosclerosis was induced in Ldlr-/- mice transplanted with Vim-/- and Vim+/+ bone marrow, and in Vim-/- and Vim+/+ mice injected with a PCSK9 gain-of-function virus. The mice were fed an atherogenic diet for 12-15 weeks. We observed impaired uptake of native LDL but increased uptake of oxLDL in Vim-/- macrophages. FACS analysis revealed increased surface expression of the scavenger receptor CD36 on Vim-/- macrophages. Vim-/- macrophages also displayed increased markers of oxidative stress, activity of the transcription factor NF-κB, secretion of proinflammatory cytokines and GLUT1-mediated glucose uptake. Vim-/- mice displayed decreased atherogenesis despite increased vascular inflammation and increased CD36 expression on macrophages in two mouse models of atherosclerosis. We demonstrate that vimentin has a strong suppressive effect on oxidative stress and that Vim-/- mice display increased vascular inflammation with increased CD36 expression on macrophages despite decreased subendothelial lipid accumulation. Thus, vimentin has a key role in regulating inflammation in macrophages during atherogenesis.
Asunto(s)
Aterosclerosis/metabolismo , Macrófagos/metabolismo , Estrés Oxidativo , Vasculitis/metabolismo , Vimentina/genética , Animales , Antígenos CD36/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/inmunología , Ratones , Ratones Transgénicos , Vimentina/metabolismoRESUMEN
The small GTPase ADP ribosylation factor 6 (ARF6) mediates endocytosis and has in addition been shown to regulate neuron differentiation. Here we investigated whether ARF6 promotes differentiation of Neuro-2a neuronal cells by modifying the cellular lipid composition. We showed that knockdown of ARF6 by siRNA in Neuro-2a cells increased neuronal outgrowth as expected. ARF6 knockdown also resulted in increased glucosylceramide levels and decreased sphingomyelin levels, but did not affect the levels of ceramide or phospholipids. We speculated that the ARF6 knockdown-induced increase in glucosylceramide was caused by an effect on glucosylceramide synthase and, in agreement, showed that ARF6 knockdown increased the mRNA levels and activity of glucosylceramide synthase. Finally, we showed that incubation of Neuro-2a cells with the glucosylceramide synthase inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) normalized the increased neuronal outgrowth induced by ARF6 knockdown. Our results thus show that ARF6 regulates neuronal differentiation through an effect on glucosylceramide synthase and glucosylceramide levels.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Diferenciación Celular/fisiología , Glucosiltransferasas/metabolismo , Neuronas/citología , Neuronas/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Diferenciación Celular/genética , Línea Celular , Glucosilceramidas/metabolismo , Glucosiltransferasas/genética , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esfingomielinas/metabolismoRESUMEN
Atherosclerotic lesions are characterized by lipid-loaded macrophages (foam cells) and hypoxic regions. Although it is well established that foam cells are produced by uptake of cholesterol from oxidized LDL, we previously showed that hypoxia also promotes foam cell formation even in the absence of exogenous lipids. The hypoxia-induced lipid accumulation results from increased triglyceride biosynthesis but the exact mechanism is unknown. Our aim was to investigate the importance of glucose in promoting hypoxia-induced de novo lipid synthesis in human macrophages. In the absence of exogenous lipids, extracellular glucose promoted the accumulation of Oil Red O-stained lipid droplets in human monocyte-derived macrophages in a concentration-dependent manner. Lipid droplet accumulation was higher in macrophages exposed to hypoxia at all assessed concentrations of glucose. Importantly, triglyceride synthesis from glucose was increased in hypoxic macrophages. GLUT3 was highly expressed in macrophage-rich and hypoxic regions of human carotid atherosclerotic plaques and in macrophages isolated from these plaques. In human monocyte-derived macrophages, hypoxia increased expression of both GLUT3 mRNA and protein, and knockdown of GLUT3 with siRNA significantly reduced both glucose uptake and lipid droplet accumulation. In conclusion, we have shown that hypoxia-induced increases in glucose uptake through GLUT3 are important for lipid synthesis in macrophages, and may contribute to foam cell formation in hypoxic regions of atherosclerotic lesions.
Asunto(s)
Células Espumosas/metabolismo , Transportador de Glucosa de Tipo 3/metabolismo , Metabolismo de los Lípidos , Lipogénesis/fisiología , Hipoxia de la Célula , Células Cultivadas , Silenciador del Gen , Glucosa/metabolismo , Transportador de Glucosa de Tipo 3/genética , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patología , Interferencia de ARNRESUMEN
Neutral lipids are stored in so-called lipid droplets, which are formed as small primordial droplets at microsomal membranes and increase in size by a fusion process. The fusion is catalyzed by the SNARE proteins SNAP23, syntaxin-5 and VAMP4. SNAP23 is involved in the insulin dependent translocation of GLUT4 to the plasma membrane, and has an important role in the development of insulin resistance. Thus fatty acids relocalize SNAP23 from the plasma membrane (and the translocation of GLUT 4) to the interior of the cell giving rise to insulin resistance. Moreover this relocalization is seen in skeletal muscles biopsies from patients with type 2 diabetes compared to matched control. Thus a missorting of SNAP23 is essential for the development of insulin resistance.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Resistencia a la Insulina , Metabolismo de los Lípidos , Lípidos , Proteínas SNARE/metabolismo , Animales , Membrana Celular/metabolismo , Membrana Celular/patología , Diabetes Mellitus Tipo 2/patología , Transportador de Glucosa de Tipo 4/metabolismo , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Transporte de Proteínas , Fracciones Subcelulares/metabolismoRESUMEN
OBJECTIVE: Our previous studies suggest that the SNARE protein synaptosomal-associated protein of 23 kDa (SNAP23) is involved in the link between increased lipid levels and insulin resistance in cardiomyocytes. The objective was to determine whether SNAP23 may also be involved in the known association between lipid accumulation in skeletal muscle and insulin resistance/type 2 diabetes in humans, as well as to identify a potential regulator of SNAP23. RESEARCH DESIGN AND METHODS: We analyzed skeletal muscle biopsies from patients with type 2 diabetes and healthy, insulin-sensitive control subjects for expression (mRNA and protein) and intracellular localization (subcellular fractionation and immunohistochemistry) of SNAP23, and for expression of proteins known to interact with SNARE proteins. Insulin resistance was determined by a euglycemic hyperinsulinemic clamp. Potential mechanisms for regulation of SNAP23 were also investigated in the skeletal muscle cell line L6. RESULTS: We showed increased SNAP23 levels in skeletal muscle from patients with type 2 diabetes compared with that from lean control subjects. Moreover, SNAP23 was redistributed from the plasma membrane to the microsomal/cytosolic compartment in the patients with the type 2 diabetes. Expression of the SNARE-interacting protein Munc18c was higher in skeletal muscle from patients with type 2 diabetes. Studies in L6 cells showed that Munc18c promoted the expression of SNAP23. CONCLUSIONS: We have translated our previous in vitro results into humans by showing that there is a change in the distribution of SNAP23 to the interior of the cell in skeletal muscle from patients with type 2 diabetes. We also showed that Munc18c is a potential regulator of SNAP23.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Resistencia a la Insulina/genética , Proteínas Munc18/metabolismo , Músculo Esquelético/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Biopsia , Glucemia/metabolismo , Citosol/metabolismo , Ambiente , Regulación de la Expresión Génica , Técnica de Clampeo de la Glucosa , Humanos , Microsomas Hepáticos/metabolismo , Proteínas Munc18/genética , Músculo Esquelético/citología , Músculo Esquelético/patología , Obesidad/complicaciones , Obesidad/genética , Obesidad/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/genética , Valores de Referencia , Gemelos MonocigóticosRESUMEN
Increased circulating free fatty acids in subjects with type 2 diabetes may contribute to activation of macrophages, and thus the development of atherosclerosis. In this study, we investigated the effect of the saturated fatty acids (SFA) palmitate, stearate, myristate and laurate, and the unsaturated fatty acid linoleate, on the production of proinflammatory cytokines in phorbol ester-differentiated THP-1 cells, a model of human macrophages. Palmitate induced secretion and mRNA expression of TNF-alpha, IL-8 and IL-1 beta, and enhanced lipopolysaccharide (LPS)-induced IL-1 beta secretion. Proinflammatory cytokine secretion was also induced by stearate, but not by the shorter chain SFA, myristate and laurate, or linoleate. Triacsin C abolished the palmitate-induced cytokine secretion, suggesting that palmitate activation to palmitoyl-CoA is required for its effect. Palmitate-induced cytokine secretion was decreased by knockdown of serine palmitoyltransferase and mimicked by C(2)-ceramide, indicating that ceramide is involved in palmitate-induced cytokine secretion. Palmitate phosphorylated p38 and JNK kinases, and blocking of these kinases with specific inhibitors diminished the palmitate-induced cytokine secretion. Palmitate also activated the AP-1 (c-Jun) transcription factor. Knockdown of MyD88 reduced the palmitate-induced IL-8, but not TNF-alpha or IL-1 beta secretion. In conclusion, our data suggest that the long-chain SFA induce proinflammatory cytokines in human macrophages via pathways involving de novo ceramide synthesis. This might contribute to the activation of macrophages in atherosclerotic plaques, especially in type 2 diabetes.
Asunto(s)
Citocinas/genética , Ácidos Grasos/farmacología , Ácido Linoleico/farmacología , Monocitos/efectos de los fármacos , Monocitos/fisiología , Línea Celular Tumoral , Ceramidas/biosíntesis , Citocinas/metabolismo , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ácidos Láuricos/farmacología , Leucemia , Monocitos/citología , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Ácido Mirístico/farmacología , Ácido Palmítico/farmacología , Palmitoil Coenzima A/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Ácidos Esteáricos/farmacología , Factor de Transcripción AP-1/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Today the WHO Growth Chart Standards, based on the growth of breastfed infants, are used. These growth curves solve the problem of the deviating observations for breastfed compared to non-breastfed infants using previous growth charts. Presently it is not clear how the mother's diet, especially the fat intake, influences the growth of the offspring. Animal experiments indicate that a low intake of n-3 polyunsaturated fatty acids via the milk may have short- and long-term negative consequences. There is limited information in man. It has been suggested that the mammary glands may have phylogenetically originated from glands providing innate immunity, later developing capacities for providing nutrition. This would agree with the fact that human milk contains so many major components which do not primarily function as nutrients, but seem to protect nutrition and growth. Lactoferrin, oligosaccharides, glycoproteins, secretory IgA antibodies, alpha-lactalbumin and the antisecretory factor have such functions.
Asunto(s)
Desarrollo Infantil , Grasas Insaturadas en la Dieta/metabolismo , Fenómenos Fisiológicos Nutricionales del Lactante/fisiología , Recién Nacido/crecimiento & desarrollo , Leche Humana/química , Alimentación con Biberón , Lactancia Materna , Femenino , Trastornos del Crecimiento/dietoterapia , Trastornos del Crecimiento/etiología , Trastornos del Crecimiento/prevención & control , Humanos , Lactante , Trastornos de la Nutrición del Lactante/dietoterapia , Trastornos de la Nutrición del Lactante/etiología , Trastornos de la Nutrición del Lactante/prevención & control , Masculino , Leche Humana/inmunologíaRESUMEN
Lactoferrin, a glycoprotein present in milk, mucosal secretions and neutrophils contributes to host defense. We have previously shown that orally given milk lactoferrin (LF) mediates anti-infectious and anti-inflammatory activities in vivo. Moreover, we have shown that LF could inhibit the LPS-induced IL-6 secretion in a human monocytic cell line, THP-1. This observation was expanded in the present study investigating the capacity of LF to inhibit cytokine mRNA expression and the involvement of nuclear transcription factor kappa B (NF-kappa B). Cells (THP-1 and Mono Mac 6 monocytic cell lines) were stimulated with Escherichia coli LPS (5-10 ng/10(6) cells) and LF was added (50-500 microg/10(6) cells) 30 min before, or after the LPS addition. By a semiquantitative RT-PCR lower levels of TNF-alpha, IL-1 beta, IL-6, and IL-8 mRNA expression were detected at the peak of the expression in THP-1 cells treated with LF. The reduction in the cytokine expression was followed by a similar reduction in the secreted cytokines as analyzed by ELISA. LF down-regulated also the IL-10 secretion (detected only in LPS-stimulated Mono Mac 6 cells). A similar level of inhibition of these cytokines was detected regardless of the time at which LF was added to the cells in relation to LPS. In addition, LF was internalized into cells and detected in the nucleoli as determined by immunostaining and immunofluorescence. Moreover, by electrophoretic mobility shift assay (EMSA) analysis LF decreased the LPS-induced binding of NF-kappa B to the TNF-alpha promoter. The results show that LF down-regulates the LPS-induced cytokine production in monocytic cells. The inhibitory mechanism is suggested to involve the interference of LF with NF-kappa B activation.