RESUMEN
BACKGROUND: Dysregulated metabolism of bioactive sphingolipids, including ceramides and sphingosine-1-phosphate, has been implicated in cardiovascular disease, although the specific species, disease contexts, and cellular roles are not completely understood. Sphingolipids are produced by the serine palmitoyltransferase enzyme, canonically composed of 2 subunits, SPTLC1 (serine palmitoyltransferase long chain base subunit 1) and SPTLC2 (serine palmitoyltransferase long chain base subunit 2). Noncanonical sphingolipids are produced by a more recently described subunit, SPTLC3 (serine palmitoyltransferase long chain base subunit 3). METHODS: The noncanonical (d16) and canonical (d18) sphingolipidome profiles in cardiac tissues of patients with end-stage ischemic cardiomyopathy and in mice with ischemic cardiomyopathy were analyzed by targeted lipidomics. Regulation of SPTLC3 by HIF1α under ischemic conditions was determined with chromatin immunoprecipitation. Transcriptomics, lipidomics, metabolomics, echocardiography, mitochondrial electron transport chain, mitochondrial membrane fluidity, and mitochondrial membrane potential were assessed in the cSPTLC3KO transgenic mice we generated. Furthermore, morphological and functional studies were performed on cSPTLC3KO mice subjected to permanent nonreperfused myocardial infarction. RESULTS: Herein, we report that SPTLC3 is induced in both human and mouse models of ischemic cardiomyopathy and leads to production of atypical sphingolipids bearing 16-carbon sphingoid bases, resulting in broad changes in cell sphingolipid composition. This induction is in part attributable to transcriptional regulation by HIF1α under ischemic conditions. Furthermore, cardiomyocyte-specific depletion of SPTLC3 in mice attenuates oxidative stress, fibrosis, and hypertrophy in chronic ischemia, and mice demonstrate improved cardiac function and increased survival along with increased ketone and glucose substrate metabolism utilization. Depletion of SPTLC3 mechanistically alters the membrane environment and subunit composition of mitochondrial complex I of the electron transport chain, decreasing its activity. CONCLUSIONS: Our findings suggest a novel essential role for SPTLC3 in electron transport chain function and a contribution to ischemic injury by regulating complex I activity.
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Cardiomiopatías , Complejo I de Transporte de Electrón , Serina C-Palmitoiltransferasa , Animales , Serina C-Palmitoiltransferasa/metabolismo , Serina C-Palmitoiltransferasa/genética , Ratones , Humanos , Cardiomiopatías/metabolismo , Cardiomiopatías/genética , Complejo I de Transporte de Electrón/metabolismo , Complejo I de Transporte de Electrón/genética , Esfingolípidos/metabolismo , Ratones Noqueados , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/genética , Isquemia Miocárdica/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Masculino , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patologíaRESUMEN
Farnesoid X receptor (FXR) activation reduces renal inflammation, but the underlying mechanisms remain elusive. Neutrophil extracellular traps (NETs) are webs of DNA formed when neutrophils undergo specialized programmed cell death (NETosis). The signaling lipid sphingosine-1-phosphate (S1P) stimulates NETosis via its receptor on neutrophils. Here, we identify FXR as a negative regulator of NETosis via repressing S1P signaling. We determined the effects of the FXR agonist obeticholic acid (OCA) in mouse models of adenosine phosphoribosyltransferase (APRT) deficiency and Alport syndrome, both genetic disorders that cause chronic kidney disease. Renal FXR activity is greatly reduced in both models, and FXR agonism reduces disease severity. Renal NETosis and sphingosine kinase 1 (Sphk1) expression are increased in diseased mice, and they are reduced by OCA in both models. Genetic deletion of FXR increases Sphk1 expression, and Sphk1 expression correlates with NETosis. Importantly, kidney S1P levels in Alport mice are two-fold higher than controls, and FXR agonism restores them back to baseline. Short-term inhibition of sphingosine synthesis in Alport mice with severe kidney disease reverses NETosis, establishing a causal relationship between S1P signaling and renal NETosis. Finally, extensive NETosis is present in human Alport kidney biopsies (six male, nine female), and NETosis severity correlates with clinical markers of kidney disease. This suggests the potential clinical relevance of the newly identified FXR-S1P-NETosis pathway. In summary, FXR agonism represses kidney Sphk1 expression. This inhibits renal S1P signaling, thereby reducing neutrophilic inflammation and NETosis.NEW & NOTEWORTHY Many preclinical studies have shown that the farnesoid X receptor (FXR) reduces renal inflammation, but the mechanism is poorly understood. This report identifies FXR as a novel regulator of neutrophilic inflammation and NETosis via the inhibition of sphingosine-1-phosphate signaling. Additionally, NETosis severity in human Alport kidney biopsies correlates with clinical markers of kidney disease. A better understanding of this signaling axis may lead to novel treatments that prevent renal inflammation and chronic kidney disease.
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Trampas Extracelulares , Nefritis , Insuficiencia Renal Crónica , Animales , Femenino , Humanos , Masculino , Ratones , Biomarcadores , Trampas Extracelulares/metabolismo , Inflamación , Insuficiencia Renal Crónica/tratamiento farmacológico , Esfingosina/metabolismoRESUMEN
Radiation-induced lung injury (RILI) is a consequence of therapeutic thoracic irradiation (TR) for many cancers, and there are no FDA-approved curative strategies. Studies report that 80% of patients who undergo TR will have CT-detectable interstitial lung abnormalities, and strategies to limit the risk of RILI may make radiotherapy less effective at treating cancer. Our lab and others have reported that lung tissue from patients with idiopathic pulmonary fibrosis (IPF) exhibits metabolic defects including increased glycolysis and lactate production. In this pilot study, we hypothesized that patients with radiation-induced lung damage will exhibit distinct changes in lung metabolism that may be associated with the incidence of fibrosis. Using liquid chromatography/tandem mass spectrometry to identify metabolic compounds, we analyzed exhaled breath condensate (EBC) in subjects with CT-confirmed lung lesions after TR for lung cancer, compared with healthy subjects, smokers, and cancer patients who had not yet received TR. The lung metabolomic profile of the irradiated group was significantly different from the three nonirradiated control groups, highlighted by increased levels of lactate. Pathway enrichment analysis revealed that EBC from the case patients exhibited concurrent alterations in lipid, amino acid, and carbohydrate energy metabolism associated with the energy-producing tricarboxylic acid (TCA) cycle. Radiation-induced glycolysis and diversion of lactate to the extracellular space suggests that pyruvate, a precursor metabolite, converts to lactate rather than acetyl-CoA, which contributes to the TCA cycle. This TCA cycle deficiency may be compensated by these alternate energy sources to meet the metabolic demands of chronic wound repair. Using an "omics" approach to probe lung disease in a noninvasive manner could inform future mechanistic investigations and the development of novel therapeutic targets.NEW & NOTEWORTHY We report that exhaled breath condensate (EBC) identifies cellular metabolic dysregulation in patients with radiation-induced lung injury. In this pilot study, untargeted metabolomics revealed a striking metabolic signature in EBC from patients with radiation-induced lung fibrosis compared to patients with lung cancer, at-risk smokers, and healthy volunteers. Patients with radiation-induced fibrosis exhibit specific changes in tricarboxylic acid (TCA) cycle energy metabolism that may be required to support the increased energy demands of fibroproliferation.
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Fibrosis Pulmonar Idiopática , Lesión Pulmonar , Neoplasias Pulmonares , Humanos , Proyectos Piloto , Fibrosis Pulmonar Idiopática/etiología , Fibrosis Pulmonar Idiopática/metabolismo , Ácido Láctico/análisis , Neoplasias Pulmonares/radioterapia , Pruebas Respiratorias/métodos , Pulmón/metabolismo , Biomarcadores/análisisRESUMEN
Obesity research has shifted in recent years to address not only the total amount of adipose tissue present in an individual but also to include adipose tissue functions such as endocrine function and thermogenesis. Data suggest that sphingolipids are critical regulators of metabolic homeostasis, and that disruption of their levels is associated with metabolic disease. Abundant data from mouse models has revealed both beneficial and deleterious roles for sphingolipids in adipose function, and numerous human studies have shown that obesity alters circulating sphingolipid profiles. Sphingolipids comprise a large family of interrelated metabolites, and pinpointing specific functions for specific lipids will be required to fully exploit the therapeutic potential of targeting sphingolipids to treat obesity and related disorders.
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Enfermedades Metabólicas , Esfingolípidos , Tejido Adiposo/metabolismo , Animales , Enfermedades Metabólicas/metabolismo , Ratones , Obesidad/metabolismo , Esfingolípidos/metabolismo , TermogénesisRESUMEN
Sphingolipids have become established participants in the pathogenesis of obesity and its associated maladies. Sphingosine kinase 1 (SPHK1), which generates S1P, has been shown to increase in liver and adipose of obese humans and mice and to regulate inflammation in hepatocytes and adipose tissue, insulin resistance, and systemic inflammation in mouse models of obesity. Previous studies by us and others have demonstrated that global sphingosine kinase 1 KO mice are protected from diet-induced obesity, insulin resistance, systemic inflammation, and NAFLD, suggesting that SPHK1 may mediate pathological outcomes of obesity. As adipose tissue dysfunction has gained recognition as a central instigator of obesity-induced metabolic disease, we hypothesized that SPHK1 intrinsic to adipocytes may contribute to HFD-induced metabolic pathology. To test this, we depleted Sphk1 from adipocytes in mice (SK1fatKO) and placed them on a HFD. In contrast to our initial hypothesis, SK1fatKO mice displayed greater weight gain on HFD and exacerbated impairment in glucose clearance. Pro-inflammatory cytokines and neutrophil content of adipose tissue were similar, as were levels of circulating leptin and adiponectin. However, SPHK1-null adipocytes were hypertrophied and had lower basal lipolytic activity. Interestingly, hepatocyte triacylglycerol accumulation and expression of pro-inflammatory cytokines and collagen 1a1 were exacerbated in SK1fatKO mice on a HFD, implicating a specific role for adipocyte SPHK1 in adipocyte function and inter-organ cross-talk that maintains overall metabolic homeostasis in obesity. Thus, SPHK1 serves a previously unidentified essential homeostatic role in adipocytes that protects from obesity-associated pathology. These findings may have implications for pharmacological targeting of the SPHK1/S1P signaling axis.
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Adipocitos/enzimología , Lipólisis , Enfermedad del Hígado Graso no Alcohólico/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficiencia , Animales , Técnicas de Inactivación de Genes , Hipertrofia , Masculino , Ratones , Enfermedad del Hígado Graso no Alcohólico/patología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genéticaRESUMEN
Saturated fatty acids (SFA) have been known to trigger inflammatory signaling in metabolic tissues; however, the effects of specific SFAs in the intestinal epithelium have not been well studied. Several previous studies have implicated disruptions in sphingolipid metabolism by oversupply of SFAs in inflammatory process. Also, our previous studies have implicated sphingosine kinase 1 (SK1) and its product sphingosine-1-phosphate (S1P) as having key roles in the regulation of inflammatory processes in the intestinal epithelium. Therefore, to define the role for specific SFAs in inflammatory responses in intestinal epithelial cells, we examined myristate (C14:0) and palmitate (C16:0). Myristate, but not palmitate, significantly induced the pro-inflammatory cytokine tumor necrosis factor α (TNFα), and it was SK1-dependent. Interestingly, myristate-induced TNFα expression was not suppressed by inhibition of S1P receptors (S1PRs), hinting at a potential novel intracellular target of S1P. Additionally, myristate regulated the expression of TNFα via JNK activation in an SK1-dependent manner, suggesting a novel S1PR-independent target as a mediator between SK1 and JNK in response to myristate. Lastly, a myristate-enriched milk fat-based diet (MFBD) increased expression of TNFα in colon tissues and elevated the S1P to sphingosine ratio, demonstrating the potential of myristate-involved pathobiologies in intestinal tissues. Taken together our studies suggest that myristate regulates the expression of TNFα in the intestinal epithelium via regulation of SK1 and JNK.
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Regulación de la Expresión Génica/efectos de los fármacos , Mucosa Intestinal/citología , Ácido Mirístico/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Factor de Necrosis Tumoral alfa/genética , Animales , Línea Celular Tumoral , Ciclooxigenasa 2/genética , Femenino , Humanos , Masculino , Ratones , RatasRESUMEN
Nonalcoholic fatty liver disease (NAFLD), a leading cause of liver dysfunction, is a metabolic disease that begins with steatosis. Sphingolipid metabolites, particularly ceramide and sphingosine-1-phosphate (S1P), have recently received attention for their potential roles in insulin resistance and hepatic steatosis. FTY720/fingolimod, a prodrug for the treatment of multiple sclerosis, is phosphorylated in vivo to its active phosphorylated form by sphingosine kinase 2 and has been shown to interfere with the actions of S1P and to inhibit ceramide biosynthesis. Therefore, in this study we investigated the effects of FTY720 in a diet-induced animal model of NAFLD (DIAMOND) that recapitulates the hallmarks of the human disease. The oral administration of FTY720 to these mice fed a high-fat diet and sugar water improved glucose tolerance and reduced steatosis. In addition to decreasing liver triglycerides, FTY720 also reduced hepatic sphingolipid levels, including ceramides, monohexosylceramides, and sphingomyelins, particularly the C16:0 and C24:1 species, as well as S1P and dihydro-S1P. FTY720 administration decreased diet-induced fatty acid synthase (FASN) expression in DIAMOND mice without affecting other key enzymes in lipogenesis. FTY720 had no effect on the expression of SREBP-1c, which transcriptionally activates FASN. However, in agreement with the notion that the active phosphorylated form of FTY720 is an inhibitor of histone deacetylases, FTY720-P accumulated in the liver, and histone H3K9 acetylation was markedly increased in these mice. Hence, FTY720 might be useful for attenuating FASN expression and triglyceride accumulation associated with steatosis.
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Dieta Alta en Grasa/efectos adversos , Clorhidrato de Fingolimod/uso terapéutico , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Organofosfatos/uso terapéutico , Esfingosina/análogos & derivados , Acetilación/efectos de los fármacos , Animales , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Femenino , Immunoblotting , Resistencia a la Insulina , Hígado/efectos de los fármacos , Hígado/metabolismo , Lisofosfolípidos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/etiología , Esfingolípidos/sangre , Esfingosina/metabolismo , Esfingosina/uso terapéutico , Triglicéridos/metabolismoRESUMEN
Saturated fatty acids (SFAs) have been shown to induce endoplasmic reticulum (ER) stress and chronic inflammatory responses, as well as alter sphingolipid metabolism. Disruptions in ER stress and sphingolipid metabolism have also been implicated in intestinal inflammation. Therefore, to elucidate the roles of SFAs in ER stress and inflammation in intestinal epithelial cells, we examined myristate (C14:0) and palmitate (C16:0). Myristate, but not palmitate, induced ER stress signaling, including activation of inositol-requiring enzyme 1 (IRE1) and X-box binding protein 1 (XBP1) signaling. Myristate significantly increased C14-ceramide levels, whereas palmitate increased several long-chain ceramides. To define the role of ceramide synthases (CerSs) in myristate-induced ER stress, we used the pharmacologic inhibitor, fumonisin B1 (FB1), and small interfering RNA (siRNA) for CerS5 and 6, the primary isoforms that are involved in C14-ceramide generation. FB1 and siRNA for CerS5 or 6 suppressed myristate-induced C14-ceramide generation and XBP1 splicing (XBP1s). Moreover, increased XBP1s induced the downstream expression of IL-6 in a CerS5/6-dependent manner. In addition, a myristate-enriched milk fat-based diet, but not a lard-based diet, increased C14-ceramide, XBP1s, and IL-6 expression in vivo. Taken together, our data suggest that myristate modulates ER stress and cytokine production in the intestinal epithelium via CerS5/6 and C14-ceramide generation.-Choi, S., Snider, J. M., Olakkengil, N., Lambert, J. M., Anderson, A. K., Ross-Evans, J. S., Cowart, L. A., Snider, A. J. Myristate-induced endoplasmic reticulum stress requires ceramide synthases 5/6 and generation of C14-ceramide in intestinal epithelial cells.
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Ceramidas/biosíntesis , Estrés del Retículo Endoplásmico/efectos de los fármacos , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Ácido Mirístico/farmacología , Esfingosina N-Aciltransferasa/metabolismo , Animales , Línea Celular , Ceramidas/genética , Células Epiteliales/patología , Femenino , Mucosa Intestinal/patología , Masculino , Ratones , Ratas , Esfingosina N-Aciltransferasa/genéticaRESUMEN
Accumulating data support a role for bioactive lipids as mediators of lipotixicity in cardiomyocytes. One class of these, the ceramides, constitutes a family of molecules that differ in structure and are synthesized by distinct enzymes, ceramide synthase (CerS)1-CerS6. Data support that specific ceramides and the enzymes that catalyze their formation play distinct roles in cell function. In a mouse model of diabetic cardiomyopathy, sphingolipid profiling revealed increases in not only the CerS5-derived ceramides but also in very long chain (VLC) ceramides derived from CerS2. Overexpression of CerS2 elevated VLC ceramides caused insulin resistance, oxidative stress, mitochondrial dysfunction, and mitophagy. Palmitate induced CerS2 and oxidative stress, mitophagy, and apoptosis, which were prevented by depletion of CerS2. Neither overexpression nor knockdown of CerS5 had any function in these processes, suggesting a chain-length dependent impact of ceramides on mitochondrial function. This concept was also supported by the observation that synthetic mitochondria-targeted ceramides led to mitophagy in a manner proportional to N-acyl chain length. Finally, blocking mitophagy exacerbated cell death. Taken together, our results support a model by which CerS2 and VLC ceramides have a distinct role in lipotoxicity, leading to mitochondrial damage, which results in subsequent adaptive mitophagy. Our data reveal a novel lipotoxic pathway through CerS2.-Law, B. A., Liao, X., Moore, K. S., Southard, A., Roddy, P., Ji, R., Szulc, Z., Bielawska, A., Schulze, P. C., Cowart, L. A. Lipotoxic very-long-chain ceramides cause mitochondrial dysfunction, oxidative stress, and cell death in cardiomyocytes.
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Ceramidas/toxicidad , Mitocondrias Cardíacas/metabolismo , Mitofagia/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular , Humanos , Mitocondrias Cardíacas/patología , Miocitos Cardíacos/patologíaRESUMEN
Bipolar disorder (BD) is a severe psychiatric illness affecting â¼1% of the world population. Valproate (VPA) and lithium, widely used for the treatment of BD, are not universally effective. These drugs have been shown to cause inositol depletion, but translating this observation to a specific therapeutic mechanism has been difficult, hampering the development of more effective therapies. We have shown previously in yeast that chronic VPA treatment induces the unfolded protein response due to increasing ceramide levels. To gain insight into the mechanisms activated during acute VPA treatment, we performed a genome-wide expression study in yeast treated with VPA for 30 min. We observed increased mRNA and protein levels of RSB1, which encodes an exporter of long chain bases dihydrosphingosine (DHS) and phytosphingosine (PHS), and further saw that VPA increased sensitivity of an rsb1Δ mutant to PHS, suggesting that VPA increases long chain base levels. Consistent with this, PHS levels were elevated in wild type and, to a greater extent, in rsb1Δ cells. Expression of ORM genes (negative regulators of PHS synthesis) and of fatty acid elongase genes FEN1 and SUR4 were decreased, and expression of YOR1 (exporter of PHS-1P) and DPL1 (lyase that degrades DHS-1P and PHS-1P) was increased. These effects were more pronounced in medium lacking inositol, and were mirrored by inositol starvation of an ino1Δ mutant. These findings provide a metabolic explanation as to how VPA-mediated inositol depletion causes increased synthesis of PHS and further support the therapeutic relevance of inositol depletion as a bipolar disorder treatment.
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Antimaníacos/farmacología , Inositol/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Esfingosina/análogos & derivados , Ácido Valproico/farmacología , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Trastorno Bipolar/tratamiento farmacológico , Ceramidas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esfingosina/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Non-alcoholic fatty liver disease (NAFLD) is a major clinical concern and its treatment consumes abundant resources. While accumulation of lipids in hepatocytes initiates the disease, this in itself is not necessarily harmful; rather, initiation of inflammation and subsequent fibrosis and cirrhosis are critical steps in NAFLD pathology. Mechanisms linking lipid overload to downstream disease progression are not fully understood; however, bioactive lipid metabolism may underlie instigation of proinflammatory signaling. With the advent of high-throughput, sensitive, and quantitative mass spectrometry-based methods for assessing lipid profiles in NAFLD, several trends have emerged, including that increases in specific sphingolipids correlate with the transition from the relatively benign condition of simple fatty liver to the much more concerning inflamed state. Continued studies that implement sphingolipid profiling will enable the extrapolations of candidate enzymes and pathways involved in NAFLD, either in biopsies or plasma from human samples, and also in animal models, from which data are much more abundant. While most data thus far are derived from targeted lipidomics approaches, unbiased, semi-quantitative approaches hold additional promise for furthering our understanding of sphingolipids as markers of and players in NAFLD.
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Metabolismo de los Lípidos , Redes y Vías Metabólicas , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Esfingolípidos/metabolismo , Animales , Progresión de la Enfermedad , Humanos , Hígado/metabolismo , Hígado/patología , Estructura Molecular , Enfermedad del Hígado Graso no Alcohólico/patología , Esfingolípidos/químicaRESUMEN
ATP-binding cassette transporters Pdr5 and Yor1 from Saccharomyces cerevisiae control the asymmetric distribution of phospholipids across the plasma membrane as well as serving as ATP-dependent drug efflux pumps. Mutant strains lacking these transporter proteins were found to exhibit very different resistance phenotypes to two inhibitors of sphingolipid biosynthesis that act either late (aureobasidin A [AbA]) or early (myriocin [Myr]) in the pathway leading to production of these important plasma membrane lipids. These pdr5Δ yor1 strains were highly AbA resistant but extremely sensitive to Myr. We provide evidence that these phenotypic changes are likely due to modulation of the plasma membrane flippase complexes, Dnf1/Lem3 and Dnf2/Lem3. Flippases act to move phospholipids from the outer to the inner leaflet of the plasma membrane. Genetic analyses indicate that lem3Δ mutant strains are highly AbA sensitive and Myr resistant. These phenotypes are fully epistatic to those seen in pdr5Δ yor1 strains. Direct analysis of AbA-induced signaling demonstrated that loss of Pdr5 and Yor1 inhibited the AbA-triggered phosphorylation of the AGC kinase Ypk1 and its substrate Orm1. Microarray experiments found that a pdr5Δ yor1 strain induced a Pdr1-dependent induction of the entire Pdr regulon. Our data support the view that Pdr5/Yor1 negatively regulate flippase function and activity of the nuclear Pdr1 transcription factor. Together, these data argue that the interaction of the ABC transporters Pdr5 and Yor1 with the Lem3-dependent flippases regulates permeability of AbA via control of plasma membrane protein function as seen for the high-affinity tryptophan permease Tat2.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Permeabilidad de la Membrana Celular/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Regulación Fúngica de la Expresión Génica , Transactivadores/metabolismoRESUMEN
Autophagy is a catabolic process involved in maintaining energy and organelle homeostasis. The relationship between obesity and the regulation of autophagy is cell type specific. Despite adverse consequences of obesity on cardiac structure and function, the contribution of altered cardiac autophagy in response to fatty acid overload is incompletely understood. Here, we report the suppression of autophagosome clearance and the activation of NADPH oxidase (Nox)2 in both high fat-fed murine hearts and palmitate-treated H9C2 cardiomyocytes (CMs). Defective autophagosome clearance is secondary to superoxide-dependent impairment of lysosomal acidification and enzyme activity in palmitate-treated CMs. Inhibition of Nox2 prevented superoxide overproduction, restored lysosome acidification and enzyme activity, and reduced autophagosome accumulation in palmitate-treated CMs. Palmitate-induced Nox2 activation was dependent on the activation of classical protein kinase Cs (PKCs), specifically PKCßII. These findings reveal a novel mechanism linking lipotoxicity with a PKCß-Nox2-mediated impairment in pH-dependent lysosomal enzyme activity that diminishes autophagic turnover in CMs.
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Autofagia/efectos de los fármacos , Grasas de la Dieta/farmacología , Lisosomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Miocitos Cardíacos/enzimología , NADPH Oxidasas/metabolismo , Ácido Palmítico/farmacología , Animales , Autofagia/genética , Línea Celular , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Lisosomas/genética , Glicoproteínas de Membrana/genética , Ratones , Miocitos Cardíacos/citología , NADPH Oxidasa 2 , NADPH Oxidasas/genética , Proteína Quinasa C beta/genética , Proteína Quinasa C beta/metabolismo , Ratas , Superóxidos/metabolismoRESUMEN
Steatohepatitis occurs in up to 20% of patients with fatty liver disease and leads to its primary disease outcomes, including fibrosis, cirrhosis, and increased risk of hepatocellular carcinoma. Mechanisms that mediate this inflammation are of major interest. We previously showed that overload of saturated fatty acids, such as that which occurs with metabolic syndrome, induced sphingosine kinase 1 (SphK1), an enzyme that generates sphingosine-1-phosphate (S1P). While data suggest beneficial roles for S1P in some contexts, we hypothesized that it may promote hepatic inflammation in the context of obesity. Consistent with this, we observed 2-fold elevation of this enzyme in livers from humans with nonalcoholic fatty liver disease and also in mice with high saturated fat feeding, which recapitulated the human disease. Mice exhibited activation of NFκB, elevated cytokine production, and immune cell infiltration. Importantly, SphK1-null mice were protected from these outcomes. Studies in cultured cells demonstrated saturated fatty acid induction of SphK1 message, protein, and activity, and also a requirement of the enzyme for NFκB signaling and increased mRNA encoding TNFα and MCP1. Moreover, saturated fat-induced NFκB signaling and elevation of TNFα and MCP1 mRNA in HepG2 cells was blocked by targeted knockdown of S1P receptor 1, supporting a role for this lipid signaling pathway in inflammation in nonalcoholic fatty liver disease.
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Ácidos Grasos/farmacología , Hepatocitos/metabolismo , Inflamación/metabolismo , Hígado/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Línea Celular , Hepatocitos/efectos de los fármacos , Humanos , Inflamación/inducido químicamente , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND & AIMS: gp96, or grp94, is an endoplasmic reticulum (ER)-localized heat shock protein 90 paralog that acts as a protein chaperone and plays an important role for example in ER homeostasis, ER stress, Wnt and integrin signaling, and calcium homeostasis, which are vital processes in oncogenesis. However, the cancer-intrinsic function of gp96 remains controversial. METHODS: We studied the roles of gp96 in liver biology in mice via an Albumin promoter-driven Cre recombinase-mediated disruption of gp96 gene, hsp90b1. The impact of gp96 status on hepatic carcinogenesis in response to diethyl-nitrosoamine (DENA) was probed. The roles of gp96 on human hepatocellular carcinoma cells (HCC) were also examined pharmacologically with a targeted gp96 inhibitor. RESULTS: We demonstrated that gp96 maintains liver development and hepatocyte function in vivo, and its loss genetically promotes adaptive accumulation of long chain ceramides, accompanied by steatotic regeneration of residual gp96+ hepatocytes. The need for compensatory expansion of gp96+ cells in the gp96- background predisposes mice to develop carcinogen-induced hepatic hyperplasia and cancer from gp96+ but not gp96- hepatocytes. We also found that genetic and pharmacological inhibition of gp96 in human HCCs perturbed multiple growth signals, and attenuated proliferation and expansion. CONCLUSIONS: gp96 is a pro-oncogenic chaperone and an attractive therapeutic target for HCC.
Asunto(s)
Carcinogénesis , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Hígado/metabolismo , Glicoproteínas de Membrana/metabolismo , Alquilantes/farmacología , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/metabolismo , Línea Celular Tumoral , Dietilnitrosamina/farmacología , Retículo Endoplásmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Ratones , Modelos Animales , Chaperonas Moleculares/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Myristate is a novel potential substrate for sphingoid base synthesis. RESULTS: Myocardial sphingoid base synthesis utilizes myristate; these sphingolipids are functionally non-redundant with canonical sphingoid bases. CONCLUSION: d16:0 and d16:1 sphingolipids constitute an appreciable proportion of cardiac dihydrosphingosine and dihydroceramide, with distinct biological roles. SIGNIFICANCE: This pool of sphingolipids may play a heretofore unsuspected role in myocardial pathology or protection. The enzyme serine palmitoyltransferase (SPT) catalyzes the formation of the sphingoid base "backbone" from which all sphingolipids are derived. Previous studies have shown that inhibition of SPT ameliorates pathological cardiac outcomes in models of lipid overload, but the metabolites responsible for these phenotypes remain unidentified. Recent in vitro studies have shown that incorporation of the novel subunit SPTLC3 broadens the substrate specificity of SPT, allowing utilization of myristoyl-coenzyme A (CoA) in addition to its canonical substrate palmitoyl-CoA. However, the relevance of these findings in vivo has yet to be determined. The present study sought to determine whether myristate-derived d16 sphingolipids are represented among myocardial sphingolipids and, if so, whether their function and metabolic routes were distinct from those of palmitate-derived d18 sphingolipids. Data showed that d16:0 sphingoid bases occurred in more than one-third of total dihydrosphingosine and dihydroceramides in myocardium, and a diet high in saturated fat promoted their de novo production. Intriguingly, d16-ceramides demonstrated highly limited N-acyl chain diversity, and in vitro enzyme activity assays showed that these bases were utilized preferentially to canonical bases by CerS1. Functional differences between myristate- and palmitate-derived sphingolipids were observed in that, unlike d18 sphingolipids and SPTLC2, d16 sphingolipids and SPTLC3 did not appear to contribute to myristate-induced autophagy, whereas only d16 sphingolipids promoted cell death and cleavage of poly(ADP-ribose) polymerase in cardiomyocytes. Thus, these results reveal a previously unappreciated component of cardiac sphingolipids with functional differences from canonical sphingolipids.
Asunto(s)
Ventrículos Cardíacos/metabolismo , Ácido Mirístico/metabolismo , Serina C-Palmitoiltransferasa/metabolismo , Esfingolípidos/metabolismo , Acilcoenzima A/metabolismo , Animales , Autofagia , Vías Biosintéticas , Gatos , Línea Celular , Supervivencia Celular , Dieta Alta en Grasa , Expresión Génica , Regulación Enzimológica de la Expresión Génica , Isoenzimas/metabolismo , Cinética , Metabolismo de los Lípidos , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/enzimología , Miocardio/citología , Miocardio/enzimología , Miocitos Cardíacos/fisiología , Palmitoil Coenzima A/metabolismo , Ratas , Serina C-Palmitoiltransferasa/genética , Esfingosina N-Aciltransferasa/metabolismo , Especificidad por SustratoRESUMEN
We previously demonstrated that sphingosine kinase 1 (Sphk1) expression and activity are up-regulated by exogenous palmitate (PAL) in a skeletal muscle model system and in diet-induced obesity in mice; however, potential functions and in vivo relevance of this have not been addressed. Here, we aimed to determine the mechanism by which PAL regulates SphK1 in muscle, and to determine potential roles for its product, sphingosine-1-phosphate (S1P), in muscle biology in the context of obesity. Cloning and analysis of the mouse Sphk1 promoter revealed a peroxisome proliferator-activated receptor (PPAR) α cis-element that mediated activation of a reporter under control of the Sphk1 promoter; direct interaction of PPARα was demonstrated by chromatin immunoprecipitation. PAL treatment induced the proinflammatory cytokine interleukin (IL)-6 in a manner dependent on SphK1, and this was attenuated by inhibition of the sphingosine-1-phosphate receptor 3 (S1PR3). Diet-induced obesity in mice demonstrated that IL-6 expression in muscle, but not adipose tissue, increased in obesity, but this was attenuated in Sphk1(-/-) mice. Moreover, plasma IL-6 levels were significantly decreased in obese Sphk1(-/-) mice relative to obese wild type mice, and muscle, but not adipose tissue IL-6 signaling was activated. These data indicate that PPARα regulates Sphk1 expression in the context of fatty acid oversupply and links PAL to muscle IL-6 production. Moreover, this function of SphK1 in diet-induced obesity suggests a potential role for SphK1 in obesity-associated pathological outcomes.
Asunto(s)
Dieta , Ácidos Grasos no Esterificados/metabolismo , Interleucina-6/biosíntesis , Músculo Esquelético/metabolismo , Obesidad/metabolismo , PPAR gamma/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transducción de Señal , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/enzimología , Obesidad/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Regiones Promotoras GenéticasRESUMEN
Liver failure secondary to metabolic dysfunction-associated steatotic liver disease (MASLD) has become the most common cause for liver transplantation in many parts of the world. Moreover, the prevalence of MASLD not only increases the demand for liver transplantation, but also limits the supply of suitable donor organs because steatosis predisposes grafts to ischemia-reperfusion injury (IRI). There are currently no pharmacological interventions to limit hepatic IRI because the mechanisms by which steatosis leads to increased injury are unclear. To identify potential novel mediators of IRI, we used liquid chromatography and mass spectrometry to assess temporal changes in the hepatic lipidome in steatotic and non-steatotic livers after warm IRI in mice. Our untargeted analyses revealed distinct differences between the steatotic and non-steatotic response to IRI and highlighted dynamic changes in lipid composition with marked changes in glycerophospholipids. These findings enhance our knowledge of the lipidomic changes that occur following IRI and provide a foundation for future mechanistic studies. A better understanding of the mechanisms underlying such changes will lead to novel therapeutic strategies to combat IRI.
Asunto(s)
Hígado Graso , Trasplante de Hígado , Daño por Reperfusión , Ratones , Animales , Lipidómica , Hígado/metabolismo , Hígado Graso/metabolismo , Trasplante de Hígado/efectos adversos , Daño por Reperfusión/metabolismo , Isquemia Tibia/efectos adversosRESUMEN
Pulmonary fibrosis is an interstitial scarring disease of the lung characterized by poor prognosis and limited treatment options. Tissue transglutaminase 2 (TG2) is believed to promote lung fibrosis by crosslinking extracellular matrix components and activating latent TGFß. This study assessed physiologic pulmonary function and metabolic alterations in the mouse bleomycin model with TG2 genetic deletion. TG2-deficient mice demonstrated attenuated the fibrosis and preservation of lung function, with significant reduction in elastance and increases in compliance and inspiratory capacity compared to control mice treated with bleomycin. Bleomycin induced metabolic changes in the mouse lung that were consistent with increased aerobic glycolysis, including increased expression of lactate dehydrogenase A and increased production of lactate, as well as increased glutamine, glutamate, and aspartate. TG2-deficient mice treated with bleomycin exhibited similar metabolic changes but with reduced magnitude. Our results demonstrate that TG2 is required for a typical fibrosis response to injury. In the absence of TG2, the fibrotic response is biochemically similar to wild-type, but lesions are smaller and lung function is preserved. We also show for the first time that profibrotic pathways of tissue stiffening and metabolic reprogramming are interconnected, and that metabolic disruptions in fibrosis go beyond glycolysis.