RESUMO
Alcohol consumption and high caloric diet are leading causes of progressive fatty liver disease. Genetic variant rs738409 in patatin-like phospholipase domain-containing protein 3 (PNPLA3 rs738409 C>G) has been repeatedly described as one of the major risk loci for alcoholic liver cirrhosis (ALC) and hepatocellular carcinoma (HCC) in humans, however, the mechanism behind this association is incompletely understood. We generated mice carrying the rs738409 variant (PNPLA3 I148M) in order to detect genotype-phenotype relationships in mice upon chow and alcohol-high fat/high sugar diet (EtOH/WD). We could clearly demonstrate that the presence of rs738409 per se is sufficient to induce spontaneous development of steatosis after 1 year in mice on a chow diet, whereas in the setting of unhealthy diet feeding, PNPLA3 I148M did not affect hepatic inflammation or fibrosis, but induced a striking lipid remodeling, microvesicular steatosis and protected from HCC formation. Using shot gun lipidomics, we detected a striking restoration of reduced long chain-polyunsaturated fatty acids (LC-PUFA)-containing TGs, docosapentaenoic acid (C22:5 n3) and omega-3-derived eicosanoids (5-HEPE, 20-HEPE, 19,20-EDP, 21-HDHA) in PNPLA3 I148M mice upon EtOH/WD. At the molecular level, PNPLA3 I148M modulated enzymes for fatty acid and TG transport and metabolism. These findings suggest (dietary) lipids as an important and independent driver of hepatic tumorigenesis. Genetic variant in PNPLA3 exerted protective effects in mice, conflicting with findings in humans. Species-related differences in physiology and metabolism should be taken into account when modeling unhealthy human lifestyle, as genetic mouse models may not always allow for translation of insight gained in humans.
Assuntos
Aciltransferases , Carcinoma Hepatocelular , Fígado Gorduroso , Neoplasias Hepáticas , Fosfolipases A2 Independentes de Cálcio , Aciltransferases/genética , Consumo de Bebidas Alcoólicas/efeitos adversos , Animais , Carcinoma Hepatocelular/genética , Ácidos Docosa-Hexaenoicos , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/genética , Predisposição Genética para Doença , Humanos , Lipase/genética , Neoplasias Hepáticas/genética , Camundongos , Fosfolipases A2 Independentes de Cálcio/genética , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Parkinson's disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein (αSyn) aggregation and associated with abnormalities in lipid metabolism. The accumulation of lipids in cytoplasmic organelles called lipid droplets (LDs) was observed in cellular models of PD. To investigate the pathophysiological consequences of interactions between αSyn and proteins that regulate the homeostasis of LDs, we used a transgenic Drosophila model of PD, in which human αSyn is specifically expressed in photoreceptor neurons. We first found that overexpression of the LD-coating proteins Perilipin 1 or 2 (dPlin1/2), which limit the access of lipases to LDs, markedly increased triacylglyclerol (TG) loaded LDs in neurons. However, dPlin-induced-LDs in neurons are independent of lipid anabolic (diacylglycerol acyltransferase 1/midway, fatty acid transport protein/dFatp) and catabolic (brummer TG lipase) enzymes, indicating that alternative mechanisms regulate neuronal LD homeostasis. Interestingly, the accumulation of LDs induced by various LD proteins (dPlin1, dPlin2, CG7900 or KlarsichtLD-BD) was synergistically amplified by the co-expression of αSyn, which localized to LDs in both Drosophila photoreceptor neurons and in human neuroblastoma cells. Finally, the accumulation of LDs increased the resistance of αSyn to proteolytic digestion, a characteristic of αSyn aggregation in human neurons. We propose that αSyn cooperates with LD proteins to inhibit lipolysis and that binding of αSyn to LDs contributes to the pathogenic misfolding and aggregation of αSyn in neurons.
Assuntos
Metabolismo dos Lipídeos/genética , Neurônios/metabolismo , Doença de Parkinson/genética , alfa-Sinucleína/genética , Animais , Animais Geneticamente Modificados/genética , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Humanos , Gotículas Lipídicas/metabolismo , Lipólise/genética , Proteínas de Membrana Transportadoras/genética , Neuroblastoma/genética , Neurônios/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Perilipina-2/genética , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia , ProteóliseRESUMO
There is little in vitro data available on long-term effects of TiO2 exposure. Such data are important for improving the understanding of underlying mechanisms of adverse health effects of TiO2. Here, we exposed pulmonary epithelial cells to two doses (0.96 and 1.92 µg/cm2) of TiO2 for 13 weeks and effects on cell cycle and cell death mechanisms, i.e., apoptosis and autophagy were determined after 4, 8 and 13 weeks of exposure. Changes in telomere length, cellular protein levels and lipid classes were also analyzed at 13 weeks of exposure. We observed that the TiO2 exposure increased the fraction of cells in G1-phase and reduced the fraction of cells in G2-phase, which was accompanied by an increase in the fraction of late apoptotic/necrotic cells. This corresponded with an induced expression of key apoptotic proteins i.e., BAD and BAX, and an accumulation of several lipid classes involved in cellular stress and apoptosis. These findings were further supported by quantitative proteome profiling data showing an increase in proteins involved in cell stress and genomic maintenance pathways following TiO2 exposure. Altogether, we suggest that cell stress response and cell death pathways may be important molecular events in long-term health effects of TiO2.
Assuntos
Células Epiteliais Alveolares/metabolismo , Titânio/efeitos adversos , Células Epiteliais Alveolares/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular , Linhagem Celular , Células Epiteliais/metabolismo , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Humanos , Pulmão/metabolismo , Nanopartículas Metálicas/efeitos adversos , Nanopartículas/efeitos adversos , Estresse Oxidativo/efeitos dos fármacos , Proteômica/métodos , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Titânio/metabolismo , Transcriptoma/genéticaRESUMO
OBJECTIVE: The rs641738C>T variant located near the membrane-bound O-acyltransferase domain containing 7 (MBOAT7) locus is associated with fibrosis in liver diseases, including non-alcoholic fatty liver disease (NAFLD), alcohol-related liver disease, hepatitis B and C. We aim to understand the mechanism by which the rs641738C>T variant contributes to pathogenesis of NAFLD. DESIGN: Mice with hepatocyte-specific deletion of MBOAT7 (Mboat7Δhep) were generated and livers were characterised by histology, flow cytometry, qPCR, RNA sequencing and lipidomics. We analysed the association of rs641738C>T genotype with liver inflammation and fibrosis in 846 NAFLD patients and obtained genotype-specific liver lipidomes from 280 human biopsies. RESULTS: Allelic imbalance analysis of heterozygous human liver samples pointed to lower expression of the MBOAT7 transcript on the rs641738C>T haplotype. Mboat7Δhep mice showed spontaneous steatosis characterised by increased hepatic cholesterol ester content after 10 weeks. After 6 weeks on a high fat, methionine-low, choline-deficient diet, mice developed increased hepatic fibrosis as measured by picrosirius staining (p<0.05), hydroxyproline content (p<0.05) and transcriptomics, while the inflammatory cell populations and inflammatory mediators were minimally affected. In a human biopsied NAFLD cohort, MBOAT7 rs641738C>T was associated with fibrosis (p=0.004) independent of the presence of histological inflammation. Liver lipidomes of Mboat7Δhep mice and human rs641738TT carriers with fibrosis showed increased total lysophosphatidylinositol levels. The altered lysophosphatidylinositol and phosphatidylinositol subspecies in MBOAT7Δhep livers and human rs641738TT carriers were similar. CONCLUSION: Mboat7 deficiency in mice and human points to an inflammation-independent pathway of liver fibrosis that may be mediated by lipid signalling and a potentially targetable treatment option in NAFLD.
Assuntos
Aciltransferases/genética , Cirrose Hepática/genética , Proteínas de Membrana/genética , Hepatopatia Gordurosa não Alcoólica/genética , Aciltransferases/deficiência , Adulto , Idoso , Animais , Biópsia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Genótipo , Haplótipos , Humanos , Inflamação/genética , Masculino , Proteínas de Membrana/deficiência , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo ÚnicoRESUMO
The effects of long-term chronic exposure of human lung cells to multi-walled carbon nanotubes (MWCNT) and their impact upon cellular proteins and lipids were investigated. Since the lung is the major target organ, an in vitro normal bronchial epithelial cell line model was used. Additionally, to better mimic exposure to manufactured nanomaterials at occupational settings, cells were continuously exposed to two non-toxic and low doses of a MWCNT for 13-weeks. MWCNT-treatment increased ROS levels in cells without increasing oxidative DNA damage and resulted in differential expression of multiple anti- and pro-apoptotic proteins. The proteomic analysis of the MWCNT-exposed cells showed that among more than 5000 identified proteins; more than 200 were differentially expressed in the treated cells. Functional analyses revealed association of these differentially regulated proteins to cellular processes such as cell death and survival, cellular assembly, and organization. Similarly, shotgun lipidomic profiling revealed accumulation of multiple lipid classes. Our results indicate that long-term MWCNT-exposure of human normal lung cells at occupationally relevant low-doses may alter both the proteome and the lipidome profiles of the target epithelial cells in the lung.
Assuntos
Metabolismo dos Lipídeos/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Nanotubos de Carbono/toxicidade , Proteoma/efeitos dos fármacos , Brônquios/metabolismo , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Proteômica , Espécies Reativas de Oxigênio/metabolismoRESUMO
Analysis of fatty acids by gas chromatography (GC) is facilitated by the generation of volatile fatty acid methyl ester (FAME) derivatives. Here we describe the esterification procedure and a typical program for separating FAMEs using a gas chromatograph equipped with a flame ionization detector or a dual stage quadrupole-mass spectrometry detector. GC is a rather simple technique that provides quantitative information on cellular fatty acid content and composition by use of an internal fatty acid standard.
Assuntos
Cromatografia Gasosa/métodos , Ácidos Graxos Voláteis/isolamento & purificação , Leveduras/química , Esterificação , Ácidos Graxos/análise , MetilaçãoRESUMO
Yeast Fld1 and Ldb16 resemble mammalian seipin, implicated in neutral lipid storage. Both proteins form a complex at the endoplasmic reticulum-lipid droplet (LD) interface. Malfunction of this complex either leads to LD clustering or to the generation of supersized LD (SLD) in close vicinity to the nuclear envelope, in response to altered phospholipid (PL) composition. We show that similar to mutants lacking Fld1, deletion of LDB16 leads to abnormal proliferation of a subdomain of the nuclear envelope, which is tightly associated with clustered LD. The human lipin-1 ortholog, the PAH1 encoded phosphatidic acid (PA) phosphatase, and its activator Nem1 are highly enriched at this site. The specific accumulation of PA-binding marker proteins indicates a local enrichment of PA in the fld1 and ldb16 mutants. Furthermore, we demonstrate that clustered LD in fld1 or ldb16 mutants are transformed to SLD if phosphatidylcholine synthesis is compromised by additional deletion of the phosphatidylethanolamine methyltransferase, Cho2. Notably, treatment of wild-type cells with oleate induced a similar LD clustering and nuclear membrane proliferation phenotype as observed in fld1 and ldb16 mutants. These data suggest that the Fld1-Ldb16 complex affects PA homeostasis at an LD-forming subdomain of the nuclear envelope. Lack of Fld1-Ldb16 leads to locally elevated PA levels that induce an abnormal proliferation of nER membrane structures and the clustering of associated LD. We suggest that the formation of SLD is a consequence of locally altered PL metabolism at this site.
Assuntos
Subunidades gama da Proteína de Ligação ao GTP/genética , Regulação Fúngica da Expressão Gênica , Proteínas Mitocondriais/genética , Membrana Nuclear/metabolismo , Ácidos Fosfatídicos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Subunidades gama da Proteína de Ligação ao GTP/deficiência , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/ultraestrutura , Metabolismo dos Lipídeos/efeitos dos fármacos , Proteínas Mitocondriais/deficiência , Mutação , Membrana Nuclear/efeitos dos fármacos , Membrana Nuclear/genética , Membrana Nuclear/ultraestrutura , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ácido Oleico/farmacologia , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolamina N-Metiltransferase/genética , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved. RESULTS: We engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER. CONCLUSIONS: We conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of biotechnological interest, such as insect cells and mammalian cells.
Assuntos
Expressão Gênica/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Fenótipo , Engenharia de ProteínasRESUMO
Numerous studies in humans link a nonsynonymous genetic polymorphism (I148M) in adiponutrin (ADPN) to various forms of fatty liver disease and liver cirrhosis. Despite its high clinical relevance, the molecular function of ADPN and the mechanism by which I148M variant affects hepatic metabolism are unclear. Here we show that ADPN promotes cellular lipid synthesis by converting lysophosphatidic acid (LPA) into phosphatidic acid. The ADPN-catalyzed LPA acyltransferase (LPAAT) reaction is specific for LPA and long-chain acyl-CoAs. Wild-type mice receiving a high-sucrose diet exhibit substantial upregulation of Adpn in the liver and a concomitant increase in LPAAT activity. In Adpn-deficient mice, this diet-induced increase in hepatic LPAAT activity is reduced. Notably, the I148M variant of human ADPN exhibits increased LPAAT activity leading to increased cellular lipid accumulation. This gain of function provides a plausible biochemical mechanism for the development of liver steatosis in subjects carrying the I148M variant.