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1.
Mol Cell ; 75(5): 1043-1057.e8, 2019 09 05.
Artículo en Inglés | MEDLINE | ID: mdl-31402097

RESUMEN

The plasma membrane (PM) is composed of a complex lipid mixture that forms heterogeneous membrane environments. Yet, how small-scale lipid organization controls physiological events at the PM remains largely unknown. Here, we show that ORP-related Osh lipid exchange proteins are critical for the synthesis of phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2], a key regulator of dynamic events at the PM. In real-time assays, we find that unsaturated phosphatidylserine (PS) and sterols, both Osh protein ligands, synergistically stimulate phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity. Biophysical FRET analyses suggest an unconventional co-distribution of unsaturated PS and phosphatidylinositol 4-phosphate (PI4P) species in sterol-containing membrane bilayers. Moreover, using in vivo imaging approaches and molecular dynamics simulations, we show that Osh protein-mediated unsaturated PI4P and PS membrane lipid organization is sensed by the PIP5K specificity loop. Thus, ORP family members create a nanoscale membrane lipid environment that drives PIP5K activity and PI(4,5)P2 synthesis that ultimately controls global PM organization and dynamics.


Asunto(s)
Proteínas Portadoras/metabolismo , Fosfatidilinositol 4,5-Difosfato/biosíntesis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Portadoras/genética , Fosfatidilinositol 4,5-Difosfato/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
2.
J Cell Sci ; 136(16)2023 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-37470177

RESUMEN

Cellular functions, such as differentiation and migration, are regulated by the extracellular microenvironment, including the extracellular matrix (ECM). Cells adhere to ECM through focal adhesions (FAs) and sense the surrounding microenvironments. Although FA proteins have been actively investigated, little is known about the lipids in the plasma membrane at FAs. In this study, we examine the lipid composition at FAs with imaging and biochemical approaches. Using the cholesterol-specific probe D4 with total internal reflection fluorescence microscopy and super-resolution microscopy, we show an enrichment of cholesterol at FAs simultaneously with FA assembly. Furthermore, we establish a method to isolate the lipid from FA-rich fractions, and biochemical quantification of the lipids reveals that there is a higher content of cholesterol and phosphatidylcholine with saturated fatty acid chains in the lipids of the FA-rich fraction than in either the plasma membrane fraction or the whole-cell membrane. These results demonstrate that plasma membrane at FAs has a locally distinct lipid composition compared to the bulk plasma membrane.


Asunto(s)
Adhesiones Focales , Fosfatidilcolinas , Adhesiones Focales/metabolismo , Fosfatidilcolinas/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Matriz Extracelular/metabolismo
3.
Hepatology ; 2024 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-38776184

RESUMEN

BACKGROUND AND AIMS: The common genetic variant rs641738 C>T is a risk factor for metabolic dysfunction-associated steatotic liver disease and metabolic dysfunction-associated steatohepatitis (MASH), including liver fibrosis, and is associated with decreased expression of the phospholipid-remodeling enzyme MBOAT7 (LPIAT1). However, whether restoring MBOAT7 expression in established metabolic dysfunction-associated steatotic liver disease dampens the progression to liver fibrosis and, importantly, the mechanism through which decreased MBOAT7 expression exacerbates MASH fibrosis remain unclear. APPROACH AND RESULTS: We first showed that hepatocyte MBOAT7 restoration in mice with diet-induced steatohepatitis slows the progression to liver fibrosis. Conversely, when hepatocyte-MBOAT7 was silenced in mice with established hepatosteatosis, liver fibrosis but not hepatosteatosis was exacerbated. Mechanistic studies revealed that hepatocyte-MBOAT7 restoration in MASH mice lowered hepatocyte-TAZ (WWTR1), which is known to promote MASH fibrosis. Conversely, hepatocyte-MBOAT7 silencing enhanced TAZ upregulation in MASH. Finally, we discovered that changes in hepatocyte phospholipids due to MBOAT7 loss-of-function promote a cholesterol trafficking pathway that upregulates TAZ and the TAZ-induced profibrotic factor Indian hedgehog (IHH). As evidence for relevance in humans, we found that the livers of individuals with MASH carrying the rs641738-T allele had higher hepatocyte nuclear TAZ, indicating higher TAZ activity and increased IHH mRNA. CONCLUSIONS: This study provides evidence for a novel mechanism linking MBOAT7-LoF to MASH fibrosis, adds new insight into an established genetic locus for MASH, and, given the druggability of hepatocyte TAZ for MASH fibrosis, suggests a personalized medicine approach for subjects at increased risk for MASH fibrosis due to inheritance of variants that lower MBOAT7.

4.
Anal Chem ; 96(29): 11771-11779, 2024 07 23.
Artículo en Inglés | MEDLINE | ID: mdl-38995673

RESUMEN

Functional mass spectrometry imaging (fMSI) is a potent tool for elucidating the spatial distribution of enzyme activities in tissues at high resolution. In this study, we applied fMSI to probe the intricate biosynthesis of phospholipids, which exist as thousands of molecular species in tissues and exhibit a unique distribution specific to cell type. By using deuterium- and 13C-labeled substrates, we visualized the activities of key enzymes involved in phospholipid synthesis, including glycerol 3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferases (LPAAT), lysophospholipid acyltransferases (LPLAT), and long-chain acyl-CoA synthetase (ACSL). Additionally, we were able to visualize a two-step sequential enzyme reaction involving ACSL and LPLAT. This novel approach unveiled significant variations in enzyme activity distribution depending on the type of fatty acids used as substrates. It will also help to reveal the mechanisms underlying the formation of numerous phospholipid species.


Asunto(s)
Espectrometría de Masas , Fosfolípidos , Fosfolípidos/metabolismo , Fosfolípidos/análisis , Animales , Ratones
5.
Arterioscler Thromb Vasc Biol ; 43(2): e66-e82, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36519468

RESUMEN

BACKGROUND: Although hypercholesterolemia reportedly counteracts lymphocyte trafficking across lymphatic vessels, the roles of lymphatic endothelial cells (LECs) in the lymphocyte regulations remain unclear. Previous studies showed that calpain-an intracellular modulatory protease-interferes with leukocyte dynamics in the blood microcirculation and is associated with hypercholesterolemic dysfunction in vascular endothelial cells. METHODS: This study investigated whether the calpain systems in LECs associate with the LEC-lymphocyte interaction under hypercholesterolemia using gene-targeted mice. RESULTS: Lipidomic analysis in hypercholesterolemic mice showed that several lysophospholipids, including lysophosphatidic acid, accumulated in the lymphatic environment. Lysophosphatidic acid enables the potentiation of calpain systems in cultured LECs, which limits their ability to stabilize regulatory T cells (Treg) without altering Th1/Th2 (T helper type1/2) subsets. This occurs via the proteolytic degradation of MEKK1 (mitogen-activated protein kinase kinase kinase 1) and the subsequent inhibition of TGF (transforming growth factor)-ß1 production in LECs. Targeting calpain systems in LECs expanded Tregs in the blood circulation and reduced aortic atherosclerosis in hypercholesterolemic mice, concomitant with the reduction of proinflammatory macrophages in the lesions. Treg expansion in the blood circulation and atheroprotection in calpain-targeted mice was prevented by the administration of TGF-ß type-I receptor inhibitor. Moreover, lysophosphatidic acid-induced calpain overactivation potentiated the IL (interleukin)-18/NF-κB (nuclear factor κB)/VCAM1 (vascular cell adhesion molecule 1) axis in LECs, thereby inhibiting lymphocyte mobility on the cells. Indeed, VCAM1 in LECs was upregulated in hypercholesterolemic mice and human cases of coronary artery disease. Neutralization of VCAM1 or targeting LEC calpain systems recovered afferent Treg transportation via lymphatic vessels in mice. CONCLUSIONS: Calpain systems in LECs have a key role in controlling Treg stability and trafficking under hypercholesterolemia.


Asunto(s)
Hipercolesterolemia , Vasos Linfáticos , Ratones , Humanos , Animales , Células Endoteliales/metabolismo , Linfocitos T Reguladores/metabolismo , Calpaína/metabolismo , Hipercolesterolemia/complicaciones , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Vasos Linfáticos/metabolismo , FN-kappa B/metabolismo
6.
Cell Mol Life Sci ; 80(6): 167, 2023 May 30.
Artículo en Inglés | MEDLINE | ID: mdl-37249637

RESUMEN

Monosialoganglioside GM3 is the simplest ganglioside involved in various cellular signaling. Cell surface distribution of GM3 is thought to be crucial for the function of GM3, but little is known about the cell surface GM3 distribution. It was shown that anti-GM3 monoclonal antibody binds to GM3 in sparse but not in confluent melanoma cells. Our model membrane study evidenced that monoclonal anti-GM3 antibodies showed stronger binding when GM3 was in less fluid membrane environment. Studies using fluorescent GM3 analogs suggested that GM3 was clustered in less fluid membrane. Moreover, fluorescent lifetime measurement showed that cell surface of high density melanoma cells is more fluid than that of low density cells. Lipidomics and fatty acid supplementation experiment suggested that monounsaturated fatty acid-containing phosphatidylcholine contributed to the cell density-dependent membrane fluidity. Our results indicate that anti-GM3 antibody senses GM3 clustering and the number and/or size of GM3 cluster differ between sparse and confluent melanoma cells.


Asunto(s)
Gangliósido G(M3) , Melanoma , Humanos , Gangliósido G(M3)/metabolismo , Membrana Celular/metabolismo , Anticuerpos Monoclonales , Melanoma/metabolismo , Recuento de Células
7.
J Lipid Res ; 64(11): 100443, 2023 11.
Artículo en Inglés | MEDLINE | ID: mdl-37714410

RESUMEN

Phosphatidylserine (PS) is an acidic phospholipid that is involved in various cellular events. Heterologous dominant mutations have been identified in the gene encoding PS synthase 1 (PSS1) in patients with a congenital disease called Lenz-Majewski syndrome (LMS). Patients with LMS show various symptoms, including craniofacial/distal-limb bone dysplasia and progressive hyperostosis. The LMS-causing gain-of-function mutants of PSS1 (PSS1LMS) have been shown to synthesize PS without control, but why the uncontrolled synthesis would lead to LMS is unknown. Here we investigated the effect of PSS1LMS on osteoclasts (OCs) to elucidate the causative mechanism of LMS. PSS1LMS did not affect the expression of OC-related genes but inhibited the formation, multinucleation, and activity of OCs. Especially, OCs expressing PSS1LMS showed abnormal patterns and dynamics of actin podosome clusters, which have roles in OC migration and fusion. PSS1LMS did not affect the level of PS but changed the acyl chain compositions of PS and phosphatidylethanolamine, and decreased the level of phosphatidylinositol. The introduction of a catalytically inactive mutation into PSSLMS canceled the changes in phospholipids and the phenotypes observed in OCs expressing PSS1LMS. A gain-of-function mutant of PSS2 (PSS2 R97K) also impaired OC formation and caused changes in phospholipid composition similar to the changes caused by PSS1LMS. Our results suggest that uncontrolled PS synthesis by PSS1LMS causes changes in the quantity or fatty acid composition of certain phospholipid classes, impairing OC formation and function, which might be a cause of osteosclerosis in patients with LMS.


Asunto(s)
Anomalías Múltiples , Discapacidad Intelectual , Humanos , Osteoclastos/metabolismo , Fosfolípidos/metabolismo
8.
J Biol Chem ; 298(1): 101470, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34890643

RESUMEN

The diversity of glycerophospholipid species in cellular membranes is immense and affects various biological functions. Glycerol-3-phosphate acyltransferases (GPATs) and lysophospholipid acyltransferases (LPLATs), in concert with phospholipase A1/2s enzymes, contribute to this diversity via selective esterification of fatty acyl chains at the sn-1 or sn-2 positions of membrane phospholipids. These enzymes are conserved across all kingdoms, and in mammals four GPATs of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family and at least 14 LPLATs, either of the AGPAT or the membrane-bound O-acyltransferase (MBOAT) families, have been identified. Here we provide an overview of the biochemical and biological activities of these mammalian enzymes, including their predicted structures, involvements in human diseases, and essential physiological roles as revealed by gene-deficient mice. Recently, the nomenclature used to refer to these enzymes has generated some confusion due to the use of multiple names to refer to the same enzyme and instances of the same name being used to refer to completely different enzymes. Thus, this review proposes a more uniform LPLAT enzyme nomenclature, as well as providing an update of recent advances made in the study of LPLATs, continuing from our JBC mini review in 2009.


Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa , Glicerofosfolípidos , Lisofosfolípidos , 1-Acilglicerofosfocolina O-Aciltransferasa/clasificación , 1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Animales , Glicerofosfolípidos/metabolismo , Humanos , Lisofosfolípidos/metabolismo , Terminología como Asunto
9.
J Lipid Res ; 63(10): 100271, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-36049524

RESUMEN

The main fatty acids at the sn-1 position of phospholipids (PLs) are saturated or monounsaturated fatty acids such as palmitic acid (C16:0), stearic acid (C18:0), and oleic acid (C18:1) and are constantly replaced, like unsaturated fatty acids at the sn-2 position. However, little is known about the molecular mechanism underlying the replacement of fatty acids at the sn-1 position, i.e., the sn-1 remodeling. Previously, we established a method to evaluate the incorporation of fatty acids into the sn-1 position of lysophospholipids (lyso-PLs). Here, we used this method to identify the enzymes capable of incorporating fatty acids into the sn-1 position of lyso-PLs (sn-1 lysophospholipid acyltransferase [LPLAT]). Screenings using siRNA knockdown and recombinant proteins for 14 LPLATs identified LPLAT7/lysophosphatidylglycerol acyltransferase 1 (LPGAT1) as a candidate. In vitro, we found LPLAT7 mainly incorporated several fatty acids into the sn-1 position of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE), with weak activities toward other lyso-PLs. Interestingly, however, only C18:0-containing phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were specifically reduced in the LPLAT7-mutant cells and tissues from knockout mice, with a concomitant increase in the level of C16:0- and C18:1-containing PC and PE. Consistent with this, the incorporation of deuterium-labeled C18:0 into PLs dramatically decreased in the mutant cells, while deuterium-labeled C16:0 and C18:1 showed the opposite dynamic. Identifying LPLAT7 as an sn-1 LPLAT facilitates understanding the biological significance of sn-1 fatty acid remodeling of PLs. We also propose to use the new nomenclature, LPLAT7, for LPGAT1 since the newly assigned enzymatic activities are quite different from the LPGAT1s previously reported.


Asunto(s)
1-Acilglicerofosfocolina O-Aciltransferasa , Fosfatidiletanolaminas , Ratones , Animales , 1-Acilglicerofosfocolina O-Aciltransferasa/genética , 1-Acilglicerofosfocolina O-Aciltransferasa/metabolismo , Lisofosfatidilcolinas , ARN Interferente Pequeño , Deuterio , Lisofosfolípidos/metabolismo , Ácidos Grasos/metabolismo , Fosfatidilcolinas/metabolismo , Ácidos Esteáricos , Ácido Palmítico/metabolismo , Ácidos Grasos Insaturados , Proteínas Recombinantes , Ácidos Oléicos , Ácidos Grasos Monoinsaturados
10.
Gut ; 70(1): 180-193, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32253259

RESUMEN

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is a common prelude to cirrhosis and hepatocellular carcinoma. The genetic rs641738 C>T variant in the lysophosphatidylinositol acyltransferase 1 (LPIAT1)/membrane bound O-acyltransferase domain-containing 7, which incorporates arachidonic acid into phosphatidylinositol (PI), is associated with the entire spectrum of NAFLD. In this study, we investigated the mechanism underlying this association in mice and cultured human hepatocytes. DESIGN: We generated the hepatocyte-specific Lpiat1 knockout mice to investigate the function of Lpiat1 in vivo. We also depleted LPIAT1 in cultured human hepatic cells using CRISPR-Cas9 systems or siRNA. The effect of LPIAT1-depletion on liver fibrosis was examined in mice fed high fat diet and in liver spheroids. Lipid species were measured using liquid chromatography-electrospray ionisation mass spectrometry. Lipid metabolism was analysed using radiolabeled glycerol or fatty acids. RESULTS: The hepatocyte-specific Lpiat1 knockout mice developed hepatic steatosis spontaneously, and hepatic fibrosis on high fat diet feeding. Depletion of LPIAT1 in cultured hepatic cells and in spheroids caused triglyceride accumulation and collagen deposition. The increase in hepatocyte fat content was due to a higher triglyceride synthesis fueled by a non-canonical pathway. Indeed, reduction in the PI acyl chain remodelling caused a high PI turnover, by stimulating at the same time PI synthesis and breakdown. The degradation of PI was mediated by a phospholipase C, which produces diacylglycerol, a precursor of triglyceride. CONCLUSION: We found a novel pathway fueling triglyceride synthesis in hepatocytes, by a direct metabolic flow of PI into triglycerides. Our findings provide an insight into the pathogenesis and therapeutics of NAFLD.


Asunto(s)
Aciltransferasas/genética , Proteínas de la Membrana/genética , Enfermedad del Hígado Graso no Alcohólico/etiología , Fosfatidilinositoles/metabolismo , Triglicéridos/metabolismo , Animales , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Metabolismo de los Lípidos , Ratones , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología
11.
J Lipid Res ; 62: 100029, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33524376

RESUMEN

Lysophosphatidic acid (LPA) is a potent signaling lipid, and state-dependent alterations in plasma LPA make it a promising diagnostic marker for various diseases. However, plasma LPA concentrations vary widely among reports, even under normal conditions. These variations can be attributed, at least in part, to the artificial metabolism of LPA after blood collection. Here, we aimed to develop an optimized plasma preparation method that reflects the concentration of LPA in the circulating blood. The main features of the devised method were suppression of both LPA production and degradation after blood collection by keeping whole blood samples at low temperature followed by the addition of an autotaxin inhibitor to plasma samples. Using this devised method, the LPA level did not change for 30 min after blood collection. Also, human and mouse LPA levels were found to be much lower than those previously reported, ranging from 40 to 50 nM with minimal variation across the individual. Finally, the increased accuracy made it possible to detect circadian rhythms in the levels of certain LPA species in mouse plasma. These results demonstrate the usefulness of the devised plasma preparation method to determine accurate plasma LPA concentrations.


Asunto(s)
Lisofosfolípidos
12.
EMBO J ; 36(12): 1719-1735, 2017 06 14.
Artículo en Inglés | MEDLINE | ID: mdl-28495679

RESUMEN

The autophagosome, a double-membrane structure mediating degradation of cytoplasmic materials by macroautophagy, is formed in close proximity to the endoplasmic reticulum (ER). However, how the ER membrane is involved in autophagy initiation and to which membrane structures the autophagy-initiation complex is localized have not been fully characterized. Here, we were able to biochemically analyze autophagic intermediate membranes and show that the autophagy-initiation complex containing ULK and FIP200 first associates with the ER membrane. To further characterize the ER subdomain, we screened phospholipid biosynthetic enzymes and found that the autophagy-initiation complex localizes to phosphatidylinositol synthase (PIS)-enriched ER subdomains. Then, the initiation complex translocates to the ATG9A-positive autophagosome precursors in a PI3P-dependent manner. Depletion of phosphatidylinositol (PI) by targeting bacterial PI-specific phospholipase C to the PIS domain impairs recruitment of downstream autophagy factors and autophagosome formation. These findings suggest that the autophagy-initiation complex, the PIS-enriched ER subdomain, and ATG9A vesicles together initiate autophagosome formation.


Asunto(s)
Autofagosomas/metabolismo , CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferasa/análisis , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/metabolismo , Biogénesis de Organelos , Animales , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia , Línea Celular , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Transporte de Proteínas
13.
J Virol ; 94(24)2020 11 23.
Artículo en Inglés | MEDLINE | ID: mdl-32999028

RESUMEN

Glycerophospholipids are major components of cell membranes. Phosphatidylethanolamine (PE) is a glycerophospholipid that is involved in multiple cellular processes, such as membrane fusion, the cell cycle, autophagy, and apoptosis. In this study, we investigated the role of PE biosynthesis in herpes simplex virus 1 (HSV-1) infection by knocking out the host cell gene encoding phosphate cytidylyltransferase 2, ethanolamine (Pcyt2), which is a key rate-limiting enzyme in one of the two major pathways for PE biosynthesis. Pcyt2 knockout reduced HSV-1 replication and caused an accumulation of unenveloped and partially enveloped nucleocapsids in the cytoplasm of an HSV-1-infected cell culture. A similar phenotype was observed when infected cells were treated with meclizine, which is an inhibitor of Pcyt2. In addition, treatment of HSV-1-infected mice with meclizine significantly reduced HSV-1 replication in the mouse brains and improved their survival rates. These results indicated that PE biosynthesis mediated by Pcyt2 was required for efficient HSV-1 envelopment in the cytoplasm of infected cells and for viral replication and pathogenicity in vivo The results also identified the PE biosynthetic pathway as a possible novel target for antiviral therapy of HSV-associated diseases and raised an interesting possibility for meclizine repositioning for treatment of these diseases, since it is an over-the-counter drug that has been used for decades against nausea and vertigo in motion sickness.IMPORTANCE Glycerophospholipids in cell membranes and virus envelopes often affect viral entry and budding. However, the role of glycerophospholipids in membrane-associated events in viral replication in herpesvirus-infected cells has not been reported to date. In this study, we have presented data showing that cellular PE biosynthesis mediated by Pcyt2 is important for HSV-1 envelopment in the cytoplasm, as well as for viral replication and pathogenicity in vivo This is the first report showing the importance of PE biosynthesis in herpesvirus infections. Our results showed that inhibition of Pcyt2, a key cell enzyme for PE synthesis, significantly inhibited HSV-1 replication and pathogenicity in mice. This suggested that the PE biosynthetic pathway, as well as the HSV-1 virion maturation pathway, can be a target for the development of novel anti-HSV drugs.


Asunto(s)
Citoplasma/virología , Herpes Simple/virología , Herpesvirus Humano 1/fisiología , Morfogénesis/fisiología , Fosfatidiletanolaminas/biosíntesis , Fosfatidiletanolaminas/fisiología , Animales , Chlorocebus aethiops , Citoplasma/metabolismo , Femenino , Células HeLa , Humanos , Ratones , Ratones Endogámicos ICR , Nucleocápside/metabolismo , ARN Nucleotidiltransferasas/genética , Células Vero , Virión/fisiología , Virulencia , Internalización del Virus , Liberación del Virus , Replicación Viral/fisiología
14.
Biochem Biophys Res Commun ; 526(1): 122-127, 2020 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-32199617

RESUMEN

Overloading of the saturated fatty acid (SFA) palmitate induces cardiomyocyte death. The purpose of this study is to elucidate signaling pathways contributing to palmitate-induced cardiomyocyte death. Palmitate-induced cardiomyocyte death was induced in Toll-like receptor 2/4 double-knockdown cardiomyocytes to a similar extent as wild-type cardiomyocytes, while cardiomyocyte death was canceled out by triacsin C, a long-chain acyl-CoA synthetase inhibitor. These results indicated that palmitate induced cytotoxicity after entry and conversion into palmitoyl-CoA. Palmitoyl-CoA is not only degraded by mitochondrial oxidation but also taken up as a component of membrane phospholipids. Palmitate overloading causes cardiomyocyte membrane fatty acid (FA) saturation, which is associated with the activation of endoplasmic reticulum (ER) unfolded protein response (UPR) signaling. We focused on the ER UPR signaling as a possible mechanism of cell death. Palmitate loading activates the UPR signal via membrane FA saturation, but not via unfolded protein overload in the ER since the chemical chaperone 4-phenylbutyrate failed to suppress palmitate-induced ER UPR. The mammalian UPR relies on three ER stress sensors named inositol requiring enzyme-1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6). Palmitate loading activated only IRE1 and PERK. Knockdown of PERK did not affect palmitate-induced cardiomyocyte death, while knockdown of IRE1 suppressed palmitate-induced cardiomyocyte death. However, knockdown of X-box binding protein 1 (XBP1), the downstream effector of IRE1, did not affect palmitate-induced cardiomyocyte death. These results were validated by pharmacological inhibitor experiments. In conclusion, we identified that palmitate-induced cardiomyocyte death was triggered by IRE1-mediated signaling independent of XBP1.


Asunto(s)
Proteínas de la Membrana/metabolismo , Miocitos Cardíacos/patología , Ácido Palmítico/toxicidad , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteína 1 de Unión a la X-Box/metabolismo , Animales , Animales Recién Nacidos , Muerte Celular/efectos de los fármacos , Células Cultivadas , Retículo Endoplásmico/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Desplegamiento Proteico/efectos de los fármacos , Ratas , Transducción de Señal/efectos de los fármacos
15.
J Mol Cell Cardiol ; 133: 1-11, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31145942

RESUMEN

BACKGROUND: The fatty acid (FA) composition of membrane phospholipid reflects at least in part dietary fat composition. Saturated FA (SFA) suppress Sirt1 activity, while monounsaturated FA (MUFA) counteract this effect. OBJECTIVE: We explored a role of Sirt1 in homeostatic control of the fatty acid composition of membrane phospholipid in the presence of SFA overload. METHODS AND RESULTS: Sirt1 deficiency in cardiomyocytes decreased the expression levels of liver X receptor (LXR)-target genes, particularly stearoyl-CoA desaturase-1 (Scd1), a rate-limiting enzyme in the cellular synthesis of MUFA from SFA, increased membrane SFA/MUFA ratio, and worsened left ventricular (LV) diastolic function in mice fed an SFA-rich high fat diet. In cultured cardiomyocytes, Sirt1 knockdown (KD) exacerbated the palmitate overload-induced increase in membrane SFA/MUFA ratio, which was associated with decrease in the expression of LXR-target genes, including Scd1. Forced overexpression of Scd1 in palmitate-overloaded Sirt1KD cardiomyocytes lowered the SFA/MUFA ratio. Nicotinamide mononucleotide (NMN) increased Sirt1 activity and Scd1 expression, thereby lowering membrane SFA/MUFA ratio in palmitate-overloaded cardiomyocytes. These effects of NMN were not observed for Scd1KD cardiomyocytes. LXRα/ßKD exacerbated palmitate overload-induced increase in membrane SFA/MUFA ratio, while LXR agonist T0901317 alleviated it. NMN failed to rescue Scd1 protein expression and membrane SFA/MUFA ratio in palmitate-overloaded LXRα/ßKD cardiomyocytes. The administration of NMN or T0901317 showed a dramatic reversal in membrane SFA/MUFA ratio and LV diastolic function in SFA-rich HFD-fed mice. CONCLUSION: Cardiac Sirt1 counteracted SFA overload-induced decrease in membrane phospholipid unsaturation and diastolic dysfunction via regulating LXR-mediated transcription of the Scd1 gene.


Asunto(s)
Diástole , Ácidos Grasos Monoinsaturados/metabolismo , Ácidos Grasos/metabolismo , Lípidos de la Membrana/metabolismo , Fosfolípidos/metabolismo , Sirtuina 1/metabolismo , Disfunción Ventricular/metabolismo , Animales , Células Cultivadas , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Metabolismo de los Lípidos , Receptores X del Hígado/agonistas , Receptores X del Hígado/metabolismo , Ratones , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Sirtuina 1/genética , Disfunción Ventricular/etiología
16.
Arch Biochem Biophys ; 663: 120-128, 2019 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-30629958

RESUMEN

BACKGROUND: Vitamin C (l-ascorbic acid, VC) and vitamin E (α-tocopherol, VE) play important physiological roles as endogenous antioxidants in many tissues and organs. However, their roles in the brain remain entirely elusive. We established senescence marker protein 30 (SMP30)/α-tocopherol transfer protein (αTTP) double knockout (DKO) mice as a novel VC and VE double-deficiency model and examined the effect of VC and VE double-deficiency on brain functions. METHODS: DKO and wild-type (WT) mice were divided into the following two groups: mice in the CE (+) group were supplied with sufficient amounts of VC and VE and mice in the CE (-) group were deficient in both VC and VE. After 8 weeks of CE (+) or CE (-) treatments, a battery of behavioral experiments was conducted to analyze cognitive functions, including memory, through the Morris water maze and Pavlovian fear conditioning tasks. RESULTS: The plasma VC and VE levels in DKO-CE (-) mice and VE level in WT-CE (-) mice were almost completely depleted after 8 weeks of the deficient treatment. The behavioral study revealed that the general behaviors, including locomotor activity and anxiety level, were not influenced by the CE (-) treatment in DKO and WT mice. However, in the Pavlovian fear conditioning task, DKO-CE (-) mice showed impaired conditioned fear memory compared with that of DKO-CE (+) mice. Furthermore, increased mRNA expression was observed in inflammatory-related genes, such as IL-6, TNFα, F4/80, and Mcp-1, in the hippocampus of DKO-CE (-) mice. CONCLUSIONS: The findings of this study provide evidence that VC and VE deficiency led to impaired conditioned fear memory possibly caused by neuroinflammation in the brain.


Asunto(s)
Deficiencia de Ácido Ascórbico/complicaciones , Encéfalo/patología , Condicionamiento Clásico , Miedo , Inflamación/complicaciones , Memoria , Deficiencia de Vitamina E/complicaciones , Animales , Ácido Ascórbico/sangre , Encéfalo/fisiopatología , Proteínas de Unión al Calcio/genética , Proteínas Portadoras/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Aprendizaje por Laberinto , Ratones , Ratones Noqueados , Vitamina E/sangre
17.
PLoS Genet ; 12(8): e1006276, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27564576

RESUMEN

Mg2+ serves as an essential cofactor for numerous enzymes and its levels are tightly regulated by various Mg2+ transporters. Here, we analyzed Caenorhabditis elegans strains carrying mutations in genes encoding cyclin M (CNNM) Mg2+ transporters. We isolated inactivating mutants for each of the five Caenorhabditis elegans cnnm family genes, cnnm-1 through cnnm-5. cnnm-1; cnnm-3 double mutant worms showed various phenotypes, among which the sterile phenotype was rescued by supplementing the media with Mg2+. This sterility was caused by a gonadogenesis defect with severely attenuated proliferation of germ cells. Using this gonadogenesis defect as an indicator, we performed genome-wide RNAi screening, to search for genes associated with this phenotype. The results revealed that RNAi-mediated inactivation of several genes restores gonad elongation, including aak-2, which encodes the catalytic subunit of AMP-activated protein kinase (AMPK). We then generated triple mutant worms for cnnm-1; cnnm-3; aak-2 and confirmed that the aak-2 mutation also suppressed the defective gonadal elongation in cnnm-1; cnnm-3 mutant worms. AMPK is activated under low-energy conditions and plays a central role in regulating cellular metabolism to adapt to the energy status of cells. Thus, we provide genetic evidence linking Mg2+ homeostasis to energy metabolism via AMPK.


Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte de Catión/genética , Ciclinas/genética , Longevidad/genética , Complejos Multiproteicos/genética , Proteínas Serina-Treonina Quinasas/genética , Serina-Treonina Quinasas TOR/genética , Proteínas Quinasas Activadas por AMP , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/metabolismo , Ciclinas/biosíntesis , Células Germinativas/crecimiento & desarrollo , Células Germinativas/metabolismo , Gónadas/crecimiento & desarrollo , Gónadas/metabolismo , Mucosa Intestinal/crecimiento & desarrollo , Mucosa Intestinal/metabolismo , Magnesio/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Familia de Multigenes/genética , Mutación , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Transducción de Señal/genética
18.
Biochem Biophys Res Commun ; 505(1): 81-86, 2018 10 20.
Artículo en Inglés | MEDLINE | ID: mdl-30241938

RESUMEN

Reelin is a secreted protein essential for the development and function of the mammalian brain. The receptors for Reelin, apolipoprotein E receptor 2 and very low-density lipoprotein receptor, belong to the low-density lipoprotein receptor family, but it is not known whether Reelin is involved in the brain lipid metabolism. In the present study, we performed lipidomic analysis of the cerebral cortex of wild-type and Reelin-deficient (reeler) mice, and found that reeler mice exhibited several compositional changes in phospholipids. First, the ratio of phospholipids containing one saturated fatty acid (FA) and one docosahexaenoic acid (DHA) or arachidonic acid (ARA) decreased. Secondly, the ratio of phospholipids containing one monounsaturated FA (MUFA) and one DHA or ARA increased. Thirdly, the ratio of phospholipids containing 5,8,11-eicosatrienoic acid, or Mead acid (MA), increased. Finally, the expression of stearoyl-CoA desaturase-1 (SCD-1) increased. As the increase of MA is seen as an index of polyunsaturated FA (PUFA) deficiency, and the expression of SCD-1 is suppressed by PUFA, these results strongly suggest that the loss of Reelin leads to PUFA deficiency. Hence, MUFA and MA are synthesized in response to this deficiency, in part by inducing SCD-1 expression. This is the first report of changes of FA composition in the reeler mouse brain and provides a basis for further investigating the new role of Reelin in the development and function of the brain.


Asunto(s)
Encéfalo/metabolismo , Moléculas de Adhesión Celular Neuronal/deficiencia , Proteínas de la Matriz Extracelular/deficiencia , Lípidos/química , Proteínas del Tejido Nervioso/deficiencia , Serina Endopeptidasas/deficiencia , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animales , Ácido Araquidónico/metabolismo , Encéfalo/embriología , Moléculas de Adhesión Celular Neuronal/genética , Ácidos Docosahexaenoicos/metabolismo , Proteínas de la Matriz Extracelular/genética , Ácidos Grasos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Metabolismo de los Lípidos , Ratones Endogámicos ICR , Ratones Mutantes Neurológicos , Proteínas del Tejido Nervioso/genética , Fosfolípidos/metabolismo , Proteína Reelina , Serina Endopeptidasas/genética , Estearoil-CoA Desaturasa/genética , Estearoil-CoA Desaturasa/metabolismo
19.
Traffic ; 16(1): 19-34, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25262571

RESUMEN

Vitamins are compounds that are essential for the normal growth, reproduction and functioning of the human body. Of the 13 known vitamins, vitamins A, D, E and K are lipophilic compounds and are therefore called fat-soluble vitamins. Because of their lipophilicity, fat-soluble vitamins are solubilized and transported by intracellular carrier proteins to exert their actions and to be metabolized properly. Vitamin A and its derivatives, collectively called retinoids, are solubilized by intracellular retinoid-binding proteins such as cellular retinol-binding protein (CRBP), cellular retinoic acid-binding protein (CRABP) and cellular retinal-binding protein (CRALBP). These proteins act as chaperones that regulate the metabolism, signaling and transport of retinoids. CRALBP-mediated intracellular retinoid transport is essential for vision in human. α-Tocopherol, the main form of vitamin E found in the body, is transported by α-tocopherol transfer protein (α-TTP) in hepatic cells. Defects of α-TTP cause vitamin E deficiency and neurological disorders in humans. Recently, it has been shown that the interaction of α-TTP with phosphoinositides plays a critical role in the intracellular transport of α-tocopherol and is associated with familial vitamin E deficiency. In this review, we summarize the mechanisms and biological significance of the intracellular transport of vitamins A and E.


Asunto(s)
Transporte Biológico/fisiología , Homeostasis/fisiología , Retinoides/metabolismo , Proteínas Celulares de Unión al Retinol/metabolismo , Vitaminas/metabolismo , Animales , Humanos , Fosfatidilinositoles/metabolismo
20.
EMBO J ; 32(9): 1265-79, 2013 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-23572076

RESUMEN

Glycerol-3-phosphate acyltransferase (GPAT) is involved in the first step in glycerolipid synthesis and is localized in both the endoplasmic reticulum (ER) and mitochondria. To clarify the functional differences between ER-GPAT and mitochondrial (Mt)-GPAT, we generated both GPAT mutants in C. elegans and demonstrated that Mt-GPAT is essential for mitochondrial fusion. Mutation of Mt-GPAT caused excessive mitochondrial fragmentation. The defect was rescued by injection of lysophosphatidic acid (LPA), a direct product of GPAT, and by inhibition of LPA acyltransferase, both of which lead to accumulation of LPA in the cells. Mitochondrial fragmentation in Mt-GPAT mutants was also rescued by inhibition of mitochondrial fission protein DRP-1 and by overexpression of mitochondrial fusion protein FZO-1/mitofusin, suggesting that the fusion/fission balance is affected by Mt-GPAT depletion. Mitochondrial fragmentation was also observed in Mt-GPAT-depleted HeLa cells. A mitochondrial fusion assay using HeLa cells revealed that Mt-GPAT depletion impaired mitochondrial fusion process. We postulate from these results that LPA produced by Mt-GPAT functions not only as a precursor for glycerolipid synthesis but also as an essential factor of mitochondrial fusion.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Mitocondrias/enzimología , Dinámicas Mitocondriales , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiología , Femenino , Eliminación de Gen , Glicerol-3-Fosfato O-Aciltransferasa/genética , Glicerol-3-Fosfato O-Aciltransferasa/fisiología , Lisofosfolípidos/metabolismo , Lisofosfolípidos/farmacología , Microsomas/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Tamaño Mitocondrial/efectos de los fármacos , Tamaño Mitocondrial/genética , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Oogénesis/genética
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