RESUMEN
Pancreatic ß cells secrete insulin in response to glucose elevation to maintain glucose homeostasis. A complex network of inter-organ communication operates to modulate insulin secretion and regulate glucose levels after a meal. Lipids obtained from diet or generated intracellularly are known to amplify glucose-stimulated insulin secretion, however, the underlying mechanisms are not completely understood. Here, we show that a Drosophila secretory lipase, Vaha (CG8093), is synthesized in the midgut and moves to the brain where it concentrates in the insulin-producing cells in a process requiring Lipid Transfer Particle, a lipoprotein originating in the fat body. In response to dietary fat, Vaha stimulates insulin-like peptide release (ILP), and Vaha deficiency results in reduced circulatory ILP and diabetic features including hyperglycemia and hyperlipidemia. Our findings suggest Vaha functions as a diacylglycerol lipase physiologically, by being a molecular link between dietary fat and lipid amplified insulin secretion in a gut-brain axis.
Asunto(s)
Encéfalo , Proteínas de Drosophila , Drosophila melanogaster , Secreción de Insulina , Insulina , Animales , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Encéfalo/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Eje Cerebro-Intestino/fisiología , Lipasa/metabolismo , Lipasa/genética , Grasas de la Dieta/metabolismo , Glucosa/metabolismo , Cuerpo Adiposo/metabolismo , Lipoproteína Lipasa/metabolismo , Lipoproteína Lipasa/genética , MasculinoRESUMEN
T helper 17 (TH17) cells are implicated in autoimmune diseases, and several metabolic processes are shown to be important for their development and function. In this study, we report an essential role for sphingolipids synthesized through the de novo pathway in TH17 cell development. Deficiency of SPTLC1, a major subunit of serine palmitoyl transferase enzyme complex that catalyzes the first and rate-limiting step of de novo sphingolipid synthesis, impaired glycolysis in differentiating TH17 cells by increasing intracellular reactive oxygen species (ROS) through enhancement of nicotinamide adenine dinucleotide phosphate oxidase 2 activity. Increased ROS leads to impaired activation of mammalian target of rapamycin C1 and reduced expression of hypoxia-inducible factor 1-alpha and c-Myc-induced glycolytic genes. SPTLCI deficiency protected mice from developing experimental autoimmune encephalomyelitis and experimental T cell transfer colitis. Our results thus show a critical role for de novo sphingolipid biosynthetic pathway in shaping adaptive immune responses with implications in autoimmune diseases.
Asunto(s)
Diferenciación Celular , Encefalomielitis Autoinmune Experimental , Serina C-Palmitoiltransferasa , Esfingolípidos , Células Th17 , Animales , Esfingolípidos/metabolismo , Esfingolípidos/biosíntesis , Células Th17/inmunología , Células Th17/metabolismo , Células Th17/citología , Ratones , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/inmunología , Serina C-Palmitoiltransferasa/metabolismo , Serina C-Palmitoiltransferasa/genética , Especies Reactivas de Oxígeno/metabolismo , Glucólisis , Ratones Noqueados , Colitis/metabolismo , Colitis/patología , Ratones Endogámicos C57BLRESUMEN
Serine palmitoyltransferase complex (SPT) mediates the first and rate-limiting step in the de novo sphingolipid biosynthetic pathway. The larger subunits SPTLC1 and SPTLC2/SPTLC3 together form the catalytic core while a smaller third subunit either SSSPTA or SSSPTB has been shown to increase the catalytic efficiency and provide substrate specificity for the fatty acyl-CoA substrates. The in vivo biological significance of these smaller subunits in mammals is still unknown. Here, using two null mutants, a conditional null for ssSPTa and a null mutant for ssSPTb, we show that SSSPTA is essential for embryogenesis and mediates much of the known functions of the SPT complex in mammalian hematopoiesis. The ssSPTa null mutants are embryonic lethal at E6.5 much like the Sptlc1 and Sptlc2 null alleles. Mx1-Cre induced deletion of ssSPTa leads to lethality and myelopoietic defect. Chimeric and competitive bone marrow transplantation experiments show that the defect in myelopoiesis is accompanied by an expansion of the Lin-Sca1+c-Kit+ stem and progenitor compartment. Progenitor cells that fail to differentiate along the myeloid lineage display evidence of endoplasmic reticulum stress. On the other hand, ssSPTb null mice are homozygous viable, and analyses of the bone marrow cells show no significant difference in the proliferation and differentiation of the adult hematopoietic compartment. SPTLC1 is an obligatory subunit for the SPT function, and because Sptlc1-/- and ssSPTa-/- mice display similar defects during development and hematopoiesis, we conclude that an SPT complex that includes SSSPTA mediates much of its developmental and hematopoietic functions in a mammalian model.
Asunto(s)
Acilcoenzima A/metabolismo , Células de la Médula Ósea/citología , Hematopoyesis/fisiología , Serina C-Palmitoiltransferasa/genética , Esfingolípidos/biosíntesis , Animales , Células de la Médula Ósea/metabolismo , Dominio Catalítico , Diferenciación Celular/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Serina C-Palmitoiltransferasa/metabolismo , Especificidad por SustratoRESUMEN
Serine palmitoyltransferase (SPT) long-chain base subunit 1 (SPTLC1) is 1 of the 2 main catalytic subunits of the SPT complex, which catalyzes the first and rate-limiting step of sphingolipid biosynthesis. Here, we show that Sptlc1 deletion in adult bone marrow (BM) cells results in defective myeloid differentiation. In chimeric mice from noncompetitive BM transplant assays, there was an expansion of the Lin- c-Kit+ Sca-1+ compartment due to increased multipotent progenitor production, but myeloid differentiation was severely compromised. We also show that defective biogenesis of sphingolipids in the endoplasmic reticulum (ER) leads to ER stress that affects myeloid differentiation. Furthermore, we demonstrate that transient accumulation of fatty acid, a substrate for sphingolipid biosynthesis, could be partially responsible for the ER stress. Independently, we find that ER stress in general, such as that induced by the chemical thapsigargin or the fatty acid palmitic acid, compromises myeloid differentiation in culture. These results identify perturbed sphingolipid metabolism as a source of ER stress, which may produce diverse pathological effects related to differential cell-type sensitivity.
Asunto(s)
Diferenciación Celular/genética , Hematopoyesis/genética , Homeostasis , Células Mieloides/citología , Células Mieloides/metabolismo , Serina C-Palmitoiltransferasa/genética , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Biología Computacional/métodos , Eliminación de Gen , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Ratones Noqueados , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/metabolismo , Bazo/citología , Bazo/metabolismoRESUMEN
The coat protein II (COPII)-coated vesicular system transports newly synthesized secretory and membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. Recruitment of cargo into COPII vesicles requires an interaction of COPII proteins either with the cargo molecules directly or with cargo receptors for anterograde trafficking. We show that cytosolic phosphatidic acid phospholipase A1 (PAPLA1) interacts with COPII protein family members and is required for the transport of Rh1 (rhodopsin 1), an N-glycosylated G protein-coupled receptor (GPCR), from the ER to the Golgi complex. In papla1 mutants, in the absence of transport to the Golgi, Rh1 is aberrantly glycosylated and is mislocalized. These defects lead to decreased levels of the protein and decreased sensitivity of the photoreceptors to light. Several GPCRs, including other rhodopsins and Bride of sevenless, are similarly affected. Our findings show that a cytosolic protein is necessary for transit of selective transmembrane receptor cargo by the COPII coat for anterograde trafficking.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila melanogaster/enzimología , Retículo Endoplásmico/enzimología , Aparato de Golgi/enzimología , Fosfolipasas A1/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Proteínas de Drosophila/química , Femenino , Masculino , Datos de Secuencia Molecular , Fosfolipasas A1/química , Transporte de ProteínasRESUMEN
Ceramide transfer protein (CERT) transfers ceramide from the endoplasmic reticulum (ER) to the Golgi complex. Its deficiency in mouse leads to embryonic death at E11.5. CERT deficient embryos die from cardiac failure due to defective organogenesis, but not due to ceramide induced apoptotic or necrotic cell death. In the current study we examined the effect of CERT deficiency in a primary cell line, namely, mouse embryonic fibroblasts (MEFs). We show that in MEFs, unlike in mutant embryos, lack of CERT does not lead to increased ceramide but causes an accumulation of hexosylceramides. Nevertheless, the defects due to defective sphingolipid metabolism that ensue, when ceramide fails to be trafficked from ER to the Golgi complex, compromise the viability of the cell. Therefore, MEFs display an incipient ER stress. While we observe that ceramide trafficking from ER to the Golgi complex is compromised, the forward transport of VSVG-GFP protein is unhindered from ER to Golgi complex to the plasma membrane. However, retrograde trafficking of the plasma membrane-associated cholera toxin B to the Golgi complex is reduced. The dysregulated sphingolipid metabolism also leads to increased mitochondrial hexosylceramide. The mitochondrial functions are also compromised in mutant MEFs since they have reduced ATP levels, have increased reactive oxygen species, and show increased glutathione reductase activity. Live-cell imaging shows that the mutant mitochondria exhibit reduced fission and fusion events. The mitochondrial dysfunction leads to an increased mitophagy in the CERT mutant MEFs. The compromised organelle function compromise cell viability and results in premature senescence of these MEFs.
Asunto(s)
Senescencia Celular/genética , Ceramidas/metabolismo , Fibroblastos/metabolismo , Mitocondrias/metabolismo , Proteínas Serina-Treonina Quinasas/deficiencia , Animales , Transporte Biológico , Proliferación Celular , Supervivencia Celular , Toxina del Cólera/metabolismo , Embrión de Mamíferos , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Femenino , Fibroblastos/patología , Expresión Génica , Aparato de Golgi/metabolismo , Metabolismo de los Lípidos/genética , Masculino , Ratones , Ratones Noqueados , Mitocondrias/patología , Cultivo Primario de Células , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Plant oils are stored in oleosomes or oil bodies, which are surrounded by a monolayer of phospholipids embedded with oleosin proteins that stabilize the structure. Recently, a structural protein, Oleosin3 (OLE3), was shown to exhibit both monoacylglycerol acyltransferase and phospholipase A(2) activities. The regulation of these distinct dual activities in a single protein is unclear. Here, we report that a serine/threonine/tyrosine protein kinase phosphorylates oleosin. Using bimolecular fluorescence complementation analysis, we demonstrate that this kinase interacts with OLE3 and that the fluorescence was associated with chloroplasts. Oleosin-green fluorescent protein fusion protein was exclusively associated with the chloroplasts. Phosphorylated OLE3 exhibited reduced monoacylglycerol acyltransferase and increased phospholipase A(2) activities. Moreover, phosphatidylcholine and diacylglycerol activated oleosin phosphorylation, whereas lysophosphatidylcholine, oleic acid, and Ca(2+) inhibited phosphorylation. In addition, recombinant peanut (Arachis hypogaea) kinase was determined to predominantly phosphorylate serine residues, specifically serine-18 in OLE3. Phosphorylation levels of OLE3 during seed germination were determined to be higher than in developing peanut seeds. These findings provide direct evidence for the in vivo substrate selectivity of the dual-specificity kinase and demonstrate that the bifunctional activities of oleosin are regulated by phosphorylation.
Asunto(s)
Aciltransferasas/metabolismo , Metabolismo de los Lípidos , Fosfolipasas A2/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Aciltransferasas/genética , Secuencia de Aminoácidos , Arachis/efectos de los fármacos , Arachis/genética , Arachis/metabolismo , Calcio/metabolismo , Cloroplastos/metabolismo , Clonación Molecular , Diglicéridos/farmacología , Genes de Plantas , Germinación , Proteínas Fluorescentes Verdes/metabolismo , Datos de Secuencia Molecular , Ácido Oléico/farmacología , Fosfatidilcolinas/farmacología , Fosfolipasas A2/genética , Fosforilación , Proteínas de Plantas/genética , Plásmidos/genética , Plásmidos/metabolismo , Mapeo de Interacción de Proteínas , Protoplastos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Semillas/metabolismo , Serina/metabolismo , Especificidad por SustratoRESUMEN
In plants, fatty oils are generally stored in spherical intracellular organelles referred to as oleosomes that are covered by proteins such as oleosin. Seeds with high oil content have more oleosin than those with low oil content. However, the exact role of oleosin in oil accumulation is thus far unclear. Here, we report the isolation of a catalytically active 14 S multiprotein complex capable of acylating monoacylglycerol from the microsomal membranes of developing peanut cotyledons. Microsomal membranes from immature peanut seeds were solubilized using 8 m urea and 10 mm CHAPS. Using two-dimensional gel electrophoresis and mass spectrometry, we identified 27 proteins in the 14 S complex. The major proteins present in the 14 S complex are conarachin, the major allergen Ara h 1, and other seed storage proteins. We identified oleosin 3 as a part of the 14 S complex, which is capable of acylating monoacylglycerol. The recombinant OLE3 microsomes from Saccharomyces cerevisiae have been shown to have both a monoacylglycerol acyltransferase and a phospholipase A(2) activity. Overexpression of the oleosin 3 (OLE3) gene in S. cerevisiae resulted in an increased accumulation of diacylglycerols and triacylglycerols and decreased phospholipids. These findings provide a direct role for a structural protein (OLE3) in the biosynthesis and mobilization of plant oils.