RESUMO
The nematode Caenorhabditis elegans requires exogenous cholesterol to survive and its depletion leads to early developmental arrest. Thus, tight regulation of cholesterol storage and distribution within the organism is critical. Previously, we demonstrated that the endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) plays a key role in C. elegans since it modulates sterol mobilization. However, the mechanism remains unknown. Here we show that mutations in the ocr-2 and osm-9 genes, coding for transient receptors potential V (TRPV) ion channels, dramatically reduce the effect of 2-AG in cholesterol mobilization. Through genetic analysis in combination with the rescue of larval arrest induced by sterol starvation, we found that the insulin/IGF-1signaling (IIS) pathway and UNC-31/CAPS, a calcium-activated regulator of neural dense-core vesicles release, are essential for 2-AG-mediated stimulation of cholesterol mobilization. These findings indicate that 2-AG-dependent cholesterol trafficking requires the release of insulin peptides and signaling through the DAF-2 insulin receptor. These results suggest that 2-AG acts as an endogenous modulator of TRPV signal transduction to control intracellular sterol trafficking through modulation of the IGF-1 signaling pathway.
Assuntos
Caenorhabditis elegans , Canabinoides , Animais , Caenorhabditis elegans/genética , Colesterol/genética , Esteróis , InsulinaRESUMO
Peroxisome proliferator-activated receptors (PPARs) play roles in glucose and lipid metabolism regulation. Pro12Ala PPAR-γ2 and +294T/C PPAR-δ have been associated with dyslipidemia, hyperglycemia and high body mass index (BMI). We compared metabolic traits and determined associations with Pro12Ala PPAR-γ2 or +294T/C PPAR-δ polymorphism among teenagers from different ethnicity. Four hundred and twelve samples with previous biochemical and biometric measurements were used. Genomic DNA from peripheral blood was extracted and analyzed by end-point PCR for Pro12Ala PPAR-γ2. The +294T/C PPAR-δ PCR product was also digested with Bsl I. Two genotype groups were formed: major allele homozygous and minor allele carriers. Pro12Ala PPAR-γ2 G minor allele frequencies were: 10% in Mestizo-1, 19% in Mestizo-2, 23% in Tarahumara, 12% in Mennonite, and 17% in the total studied population. The +294T/C PPAR-δ C minor allele frequencies were: 18% in Mestizo-1, 20% in Mestizo-2, 6% in Tarahumara, 13% in Mennonite, and 12% in the total studied population. Teenagers with PPAR-γ2 G allele showed a greater risk for either high waist/height ratio or low high-density lipoprotein; and, also had lower total cholesterol. Whereas, PPAR-γ2 G allele showed lower overweight/obesity phenotype (BMI Z-score) frequency, PPAR-δ C allele was a risk factor for it. Metabolic traits were associated with both PPAR polymorphisms.
Assuntos
Peso Corporal/genética , Colesterol/genética , Lipoproteínas HDL/genética , PPAR delta/genética , PPAR gama/genética , Polimorfismo de Nucleotídeo Único , Adolescente , Colesterol/sangue , Feminino , Frequência do Gene , Humanos , Lipoproteínas HDL/sangue , Masculino , México , Mutação de Sentido IncorretoRESUMO
Liver X Receptors (LXRs) are ligand dependent transcription factors activated by oxidized cholesterol metabolites (oxysterols) that play fundamental roles in the transcriptional control of lipid metabolism, cholesterol transport and modulation of inflammatory responses. In the last decade, LXRs have become attractive pharmacological targets for intervention in human metabolic diseases and thus, several efforts have concentrated on the development of synthetic analogues able to modulate LXR transcriptional response. In this sense, we have previously found that cholestenoic acid analogues with a modified side chain behave as LXR inverse agonists. To further investigate the structure-activity relationships and to explore how cholestenoic acid derivatives interact with the LXRs, we evaluated the LXR biological activity of new analogues containing a C24-C25 double bond. Furthermore, a microarray assay was performed to evaluate the recruitment of coregulators to recombinant LXR LBD upon ligand binding. Also, conventional and accelerated molecular dynamics simulations were applied to gain insight on the molecular determinants involved in the inverse agonism. As LXR inverse agonists emerge as very promising candidates to control LXR activity, the cholestenoic acid analogues here depicted constitute a new relevant steroidal scaffold to inhibit LXR action.
Assuntos
Colestenos/farmacologia , Colesterol/metabolismo , Receptores X do Fígado/química , Oxisteróis/metabolismo , Colestenos/química , Colesterol/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Metabolismo dos Lipídeos , Receptores X do Fígado/genética , Receptores X do Fígado/ultraestrutura , Análise em Microsséries , Conformação Molecular , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Oxisteróis/química , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Male EBP disorder with neurologic defects (MEND) syndrome is an X-linked disease caused by hypomorphic mutations in the EBP (emopamil-binding protein) gene. Modifier genes may explain the clinical variability among individuals who share a primary mutation. METHODS: We studied four males (Patient 1 to Patient 4) exhibiting a descending degree of phenotypic severity from a family with MEND syndrome. To identify candidate modifier genes that explain the phenotypic variability, variants of homeostasis cholesterol genes identified by whole-exome sequencing (WES) were ranked according to the predicted magnitude of their effect through an in-house scoring system. RESULTS: Twenty-seven from 105 missense variants found in 45 genes of the four exomes were considered significant (-5 to -9 scores). We found a direct genotype-phenotype association based on the differential accumulation of potentially functional gene variants among males. Patient 1 exhibited 17 variants, both Patients 2 and 3 exhibited nine variants, and Patient 4 exhibited only five variants. CONCLUSION: We conclude that APOA5 (rs3135506), ABCA1 (rs9282541), and APOB (rs679899 and rs12714225) are the most relevant candidate modifier genes in this family. Relative accumulation of the deficiencies associated with variants of these genes along with other lesser deficiencies in other genes appears to explain the variable expressivity in MEND syndrome.
Assuntos
Transportador 1 de Cassete de Ligação de ATP , Apolipoproteína A-V , Apolipoproteína B-100 , Colesterol , Exoma , Polimorfismo Genético , Síndrome de Waardenburg , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Apolipoproteína A-V/genética , Apolipoproteína A-V/metabolismo , Apolipoproteína B-100/genética , Apolipoproteína B-100/metabolismo , Colesterol/genética , Colesterol/metabolismo , Feminino , Estudos de Associação Genética , Homeostase/genética , Humanos , Masculino , Fenótipo , Índice de Gravidade de Doença , Síndrome de Waardenburg/genética , Síndrome de Waardenburg/metabolismo , Síndrome de Waardenburg/patologiaRESUMO
Macrophages play an essential role in lipid metabolism; however, the excessive uptake of modified lipids and cholesterol crystals (CC) leads to the formation of pro-inflammatory lipid-laden macrophages called foam cells. Since the α-1,6- and ß-1,3-d-glucans from the basidiome and the mycelium of the edible mushroom Pleurotus albidus have previously been shown to regulate macrophage function, these glucans were tested in macrophage-like THP-1 cells previously exposed to acetylated low-density lipoproteins (acLDL) or CC. The glucans inhibited lipid-induced inflammation, but only the ß-1,3-d-glucan regulated both the NLRP3 inflammasome activation and the expression of genes involved on lipid efflux in acLDL- or CC-pretreated cells, thereby reducing foam cell formation. In contrast, the two α-1,6-glucans tested inhibited foam cell formation only in acLDL-pretreated cells and had no effect on the expression of the peroxisome proliferator-activated receptor gamma and liver X receptor alpha genes, suggesting that these glucans regulate lipid influx rather than lipid efflux. Thus, α- and ß-d-glucans differentially regulate lipid-induced inflammation and foam cell formation in macrophage-like cells. Furthermore, results emphasize that P. albidus has potential to be used as a functional food or as a source for the extraction of biologically-active glucans.
Assuntos
Células Espumosas/efeitos dos fármacos , Glucanos/farmacologia , Inflamação/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Linhagem Celular , Colesterol/genética , Células Espumosas/química , Expressão Gênica/efeitos dos fármacos , Glucanos/química , Glucanos/isolamento & purificação , Humanos , Inflamação/genética , Inflamação/patologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/química , Lipoproteínas LDL/genética , Receptores X do Fígado/genética , Macrófagos/metabolismo , Micélio/química , Micélio/efeitos dos fármacos , PPAR gama/genética , Pleurotus/químicaRESUMO
The TMEM16A-mediated Ca2+-activated Cl- current drives several important physiological functions. Membrane lipids regulate ion channels and transporters but their influence on members of the TMEM16 family is poorly understood. Here we have studied the regulation of TMEM16A by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), cholesterol, and fatty acids using patch clamp, biochemistry and fluorescence microscopy. We found that depletion of membrane PI(4,5)P2 causes a decline in TMEM16A current that is independent of cytoskeleton, but is partially prevented by removing intracellular Ca2+. On the other hand, supplying PI(4,5)P2 to inside-out patches attenuated channel rundown and/or partially rescued activity after channel rundown. Also, depletion (with methyl-ß-cyclodextrin M-ßCD) or restoration (with M-ßCD+cholesterol) of membrane cholesterol slows down the current decay observed after reduction of PI(4,5)P2. Neither depletion nor restoration of cholesterol change PI(4,5)P2 content. However, M-ßCD alone transiently increases TMEM16A activity and dampens rundown whereas M-ßCD+cholesterol increases channel rundown. Thus, PI(4,5)P2 is required for TMEM16A function while cholesterol directly and indirectly via a PI(4,5)P2-independent mechanism regulate channel function. Stearic, arachidonic, oleic, docosahexaenoic, and eicosapentaenoic fatty acids as well as methyl stearate inhibit TMEM16A in a dose- and voltage-dependent manner. Phosphatidylserine, a phospholipid whose hydrocarbon tails contain stearic and oleic acids also inhibits TMEM16A. Finally, we show that TMEM16A remains in the plasma membrane after treatment with M-ßCD, M-ßCD+cholesterol, oleic, or docosahexaenoic acids. Thus, we propose that lipids and fatty acids regulate TMEM16A channels through a membrane-delimited protein-lipid interaction.
Assuntos
Anoctamina-1/metabolismo , Sinalização do Cálcio/fisiologia , Membrana Celular/metabolismo , Colesterol/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Anoctamina-1/genética , Cálcio/metabolismo , Membrana Celular/genética , Colesterol/genética , Ácidos Graxos/genética , Células HEK293 , Humanos , Proteínas de Neoplasias/genética , Fosfatidilinositol 4,5-Difosfato/genéticaRESUMO
Previous studies have shown that cytokines can affect serum lipoprotein concentrations. The aim of this study was to examine the association between IL-10 gene polymorphisms and serum lipoprotein levels of Han Chinese individuals. A total of 359 Han Chinese people were enrolled in this investigation. IL-10 -592, -819, and -1082 genotypes were established using polymerase chain reaction-restriction fragment length polymorphism analysis. An automatic biochemistry analyzer was used to determine serum concentrations of total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and very low-density lipoprotein (VLDL) in each individual. We observed that the three IL-10 polymorphisms did not significantly differ in terms of age or age of carrier (P > 0.05), and the -592 and -819 variants did not significantly affect serum lipoprotein levels (P > 0.05). HDL concentrations were higher and TG levels were lower in carriers of the -1082 GA genotype compared to those with the AA genotype, and these differences were statistically significant (P < 0.05). However, TC, VLDL, and LDL levels were unaffected by this sequence variation (P > 0.05). Our results suggest that the polymorphism at position -1082 in the promoter region of IL-10 may affect serum HDL and TG concentrations, while other variants of this gene appear to have no relationship with serum lipoprotein levels.
Assuntos
Interleucina-10/genética , Lipoproteínas HDL/sangue , Lipoproteínas LDL/sangue , Lipoproteínas VLDL/sangue , Adulto , Idoso , Colesterol/sangue , Colesterol/genética , Feminino , Genótipo , Humanos , Interleucina-10/sangue , Lipoproteínas HDL/genética , Lipoproteínas LDL/genética , Lipoproteínas VLDL/genética , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Triglicerídeos/sangue , Triglicerídeos/genéticaRESUMO
BACKGROUND: Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder characterized by the accumulation of free cholesterol in lysosomes. There are currently no effective FDA-approved treatments for NPC, although in the last years the inhibition of histone deacetylases (HDACs) has emerged as a potential treatment for this disease. However, the molecular mechanisms that deregulate HDAC activity in NPC disease are unknown. Previously our group had shown that the proapoptotic tyrosine kinase c-Abl signaling is activated in NPC neurons. Here, we demonstrate that c-Abl activity increases HDAC2 levels inducing neuronal gene repression of key synaptic genes in NPC models. RESULTS: Our data show that: i) HDAC2 levels and activity are increased in NPC neuronal models and in Npc1(-/-) mice; ii) inhibition of c-Abl or c-Abl deficiency prevents the increase of HDAC2 protein levels and activity in NPC neuronal models; iii) c-Abl inhibition decreases the levels of HDAC2 tyrosine phosphorylation; iv) treatment with methyl-ß-cyclodextrin and vitamin E decreases the activation of the c-Abl/HDAC2 pathway in NPC neurons; v) in vivo treatment with two c-Abl inhibitors prevents the increase of HDAC2 protein levels in the brain of Npc1(-/-) mice; and vi) c-Abl inhibition prevents HDAC2 recruitment to the promoter of neuronal genes, triggering an increase in their expression. CONCLUSION: Our data show the involvement of the c-Abl/HDAC2 signaling pathway in the regulation of neuronal gene expression in NPC neuronal models. Thus, inhibition of c-Abl could be a pharmacological target for preventing the deleterious effects of increased HDAC2 levels in NPC disease.
Assuntos
Histona Desacetilase 2/genética , Neurônios/metabolismo , Doença de Niemann-Pick Tipo C/genética , Proteínas Proto-Oncogênicas c-abl/genética , Animais , Colesterol/genética , Colesterol/metabolismo , Ciclodextrinas/administração & dosagem , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Histona Desacetilase 2/biossíntese , Humanos , Lisossomos/metabolismo , Camundongos , Neurônios/patologia , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Doença de Niemann-Pick Tipo C/patologia , Proteínas Proto-Oncogênicas c-abl/biossíntese , Transdução de Sinais/efeitos dos fármacos , Vitamina E/administração & dosagemRESUMO
Atherosclerosis has been associated with mitochondria dysfunction and damage. Our group demonstrated previously that hypercholesterolemic mice present increased mitochondrial reactive oxygen (mtROS) generation in several tissues and low NADPH/NADP+ ratio. Here, we investigated whether spontaneous atherosclerosis in these mice could be modulated by treatments that replenish or spare mitochondrial NADPH, named citrate supplementation, cholesterol synthesis inhibition, or both treatments simultaneously. Robust statistical analyses in pooled group data were performed in order to explain the variation of atherosclerosis lesion areas as related to the classic atherosclerosis risk factors such as plasma lipids, obesity, and oxidative stress, including liver mtROS. Using three distinct statistical tools (univariate correlation, adjusted correlation, and multiple regression) with increasing levels of stringency, we identified a novel significant association and a model that reliably predicts the extent of atherosclerosis due to variations in mtROS. Thus, results show that atherosclerosis lesion area is positively and independently correlated with liver mtROS production rates. Based on these findings, we propose that modulation of mitochondrial redox state influences the atherosclerosis extent.
Assuntos
Aterosclerose/metabolismo , Colesterol/metabolismo , Mitocôndrias Hepáticas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/patologia , Colesterol/genética , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/genética , Mitocôndrias Hepáticas/patologia , NADP/genética , NADP/metabolismoRESUMO
Advanced glycation end products (AGE) are elevated in diabetes mellitus (DM) and predict the development of atherosclerosis. AGE-albumin induces oxidative stress, which is linked to a reduction in ABCA-1 and cholesterol efflux. We characterized the glycation level of human serum albumin (HSA) isolated from poorly controlled DM2 (n = 11) patients compared with that of control (C, n = 12) individuals and determined the mechanism by which DM2-HSA can interfere in macrophage lipid accumulation. The HSA glycation level was analyzed by MALDI/MS. Macrophages were treated for 18 h with C- or DM2-HSA to measure the (14) C-cholesterol efflux, the intracellular lipid accumulation and the cellular ABCA-1 protein content. Agilent arrays (44000 probes) were used to analyze gene expression, and the differentially expressed genes were validated by real-time RT-PCR. An increased mean mass was observed in DM2-HSA compared with C-HSA, reflecting the condensation of at least 5 units of glucose. The cholesterol efflux mediated by apo AI, HDL3 , and HDL2 was impaired in DM2-HSA-treated cells, which was related to greater intracellular lipid accumulation. DM2-HSA decreased Abcg1 mRNA expression by 26%. Abca1 mRNA was unchanged, although the final ABCA-1 protein content decreased. Compared with C-HAS-treated cells, NADPH oxidase 4 mRNA expression increased in cells after DM2-HSA treatment. Stearoyl-Coenzyme A desaturase 1, janus kinase 2, and low density lipoprotein receptor mRNAs were reduced by DM2-HSA. The level of glycation that occurs in vivo in DM2-HSA-treated cells selectively alters macrophage gene expression, impairing cholesterol efflux and eliciting intracellular lipid accumulation, which contribute to atherogenesis, in individuals with DM2.