RESUMEN
Studies in vitro have suggested that both sterol carrier protein-2/sterol carrier protein-x (Scp-2/Scp-x) and liver fatty acid binding protein [Fabp1 (L-FABP)] gene products facilitate hepatic uptake and metabolism of lipotoxic dietary phytol. However, interpretation of physiological function in mice singly gene ablated in the Scp-2/Scp-x has been complicated by concomitant upregulation of FABP1. The work presented herein provides several novel insights: i) An 8-anilino-1-naphthalenesulfonic acid displacement assay showed that neither SCP-2 nor L-FABP bound phytol, but both had high affinity for its metabolite, phytanic acid; ii) GC-MS studies with phytol-fed WT and Fabp1/Scp-2/SCP-x gene ablated [triple KO (TKO)] mice showed that TKO exacerbated hepatic accumulation of phytol metabolites in vivo in females and less so in males. Concomitantly, dietary phytol increased hepatic levels of total long-chain fatty acids (LCFAs) in both male and female WT and TKO mice. Moreover, in both WT and TKO female mice, dietary phytol increased hepatic ratios of saturated/unsaturated and polyunsaturated/monounsaturated LCFAs, while decreasing the peroxidizability index. However, in male mice, dietary phytol selectively increased the saturated/unsaturated ratio only in TKO mice, while decreasing the peroxidizability index in both WT and TKO mice. These findings suggested that: 1) SCP-2 and FABP1 both facilitated phytol metabolism after its conversion to phytanic acid; and 2) SCP-2/SCP-x had a greater impact on hepatic phytol metabolism than FABP1.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Unión a Ácidos Grasos/deficiencia , Proteínas de Unión a Ácidos Grasos/genética , Técnicas de Inactivación de Genes , Hígado/metabolismo , Fitol/metabolismo , Animales , Retículo Endoplásmico/metabolismo , Femenino , Masculino , Ratones , Peroxisomas/metabolismo , Ácido Fitánico/metabolismo , Especificidad por SustratoRESUMEN
While prior studies focusing on male mice suggest a role for sterol carrier protein-2/sterol carrier protein-x (SCP-2/SCP-x; DKO) on hepatic phytol metabolism, its role in females is unresolved. This issue was addressed using female and male wild-type (WT) and DKO mice fed a phytoestrogen-free diet without or with 0.5% phytol. GC/MS showed that hepatic: i) phytol was absent and its branched-chain fatty acid (BCFA) metabolites were barely detectable in WT control-fed mice; ii) accumulation of phytol as well as its peroxisomal metabolite BCFAs (phytanic acid ¼ pristanic and 2,3-pristenic acids) was increased by dietary phytol in WT females, but only slightly in WT males; iii) accumulation of phytol and BCFA was further increased by DKO in phytol-fed females, but much more markedly in males. Livers of phytol-fed WT female mice as well as phytol-fed DKO female and male mice also accumulated increased proportion of saturated straight-chain fatty acids (LCFA) at the expense of unsaturated LCFA. Liver phytol accumulation was not due to increased SCP-2 binding/transport of phytol since SCP-2 bound phytanic acid, but not its precursor phytol. Thus, the loss of Scp-2/Scp-x contributed to a sex-dependent hepatic accumulation of dietary phytol and BCFA.
Asunto(s)
Proteínas Portadoras/metabolismo , Metabolismo de los Lípidos/fisiología , Hígado/metabolismo , Ácido Fitánico/análogos & derivados , Fitol/farmacocinética , Administración Oral , Animales , Proteínas Portadoras/genética , Femenino , Silenciador del Gen , Masculino , Tasa de Depuración Metabólica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ácido Fitánico/metabolismo , Fitol/administración & dosificación , Factores SexualesRESUMEN
Although expression of the human liver fatty acid binding protein (FABP1) T94A variant alters serum lipoprotein cholesterol levels in human subjects, nothing is known whereby the variant elicits these effects. This issue was addressed by in vitro cholesterol binding assays using purified recombinant wild-type (WT) FABP1 T94T and T94A variant proteins and in cultured primary human hepatocytes expressing the FABP1 T94T (genotyped as TT) or T94A (genotyped as CC) proteins. The human FABP1 T94A variant protein had 3-fold higher cholesterol-binding affinity than the WT FABP1 T94T as shown by NBD-cholesterol fluorescence binding assays and by cholesterol isothermal titration microcalorimetry (ITC) binding assays. CC variant hepatocytes also exhibited 30% higher total FABP1 protein. HDL- and LDL-mediated NBD-cholesterol uptake was faster in CC variant than TT WT human hepatocytes. VLDL-mediated uptake of NBD-cholesterol did not differ between CC and TT human hepatocytes. The increased HDL- and LDL-mediated NBD-cholesterol uptake was not associated with any significant change in mRNA levels of SCARB1, LDLR, CETP, and LCAT encoding the key proteins in lipoprotein cholesterol uptake. Thus, the increased HDL- and LDL-mediated NBD-cholesterol uptake by CC hepatocytes may be associated with higher affinity of T94A protein for cholesterol and/or increased total T94A protein level.
Asunto(s)
Colesterol/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Hepatocitos/metabolismo , Mutación Missense , Alanina/genética , Sustitución de Aminoácidos , Animales , Transporte Biológico/genética , Células Cultivadas , Proteínas de Unión a Ácidos Grasos/metabolismo , Femenino , Humanos , Metabolismo de los Lípidos/genética , Persona de Mediana Edad , Treonina/genéticaRESUMEN
Although liver fatty acid binding protein (L-FABP) binds fibrates and PPARα in vitro and enhances fibrate induction of PPARα in transformed cells, the functional significance of these findings is unclear, especially in normal hepatocytes. Studies with cultured primary mouse hepatocytes show that: 1) At physiological (6mM) glucose, fibrates (bezafibrate, fenofibrate) only weakly activated PPARα transcription of genes in LCFA ß-oxidation; 2) High (11-20mM) glucose, but not maltose (osmotic control), significantly potentiated fibrate-induction of mRNA of these and other PPARα target genes to increase LCFA ß-oxidation. These effects were associated with fibrate-mediated redistribution of L-FABP into nuclei-an effect prolonged by high glucose-but not with increased de novo fatty acid synthesis from glucose; 3) Potentiation of bezafibrate action by high glucose required an intact L-FABP/PPARα signaling pathway as shown with L-FABP null, PPARα null, PPARα inhibitor-treated WT, or PPARα-specific fenofibrate-treated WT hepatocytes. High glucose alone in the absence of fibrate was ineffective. Thus, high glucose potentiation of PPARα occurred through FABP/PPARα rather than indirectly through other PPARs or glucose induced signaling pathways. These data indicated L-FABP's importance in fibrate-induction of hepatic PPARα LCFA ß-oxidative genes, especially in the context of high glucose levels.
Asunto(s)
Bezafibrato/farmacología , Proteínas de Unión a Ácidos Grasos/metabolismo , Fenofibrato/farmacología , Glucosa/farmacología , Hepatocitos/metabolismo , Hipolipemiantes/farmacología , PPAR alfa/metabolismo , Edulcorantes/farmacología , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Ácidos Grasos/genética , Ácidos Grasos/metabolismo , Hepatocitos/citología , Ratones , Ratones Mutantes , Oxidación-Reducción/efectos de los fármacos , PPAR alfa/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
On the basis of their abilities to bind bile acids and/or cholesterol, the physiological role(s) of liver fatty acid-binding protein (L-FABP) and sterol carrier protein (SCP) 2/SCP-x (SCP-2/SCP-x) gene products in biliary bile acid and cholesterol formation was examined in gene-ablated male mice. L-FABP (LKO) or L-FABP/SCP-2/SCP-x [triple-knockout (TKO)] ablation markedly decreased hepatic bile acid concentration, while SCP-2/SCP-x [double-knockout (DKO)] ablation alone had no effect. In contrast, LKO increased biliary bile acid, while DKO and TKO had no effect on biliary bile acid levels. LKO and DKO also altered biliary bile acid composition to increase bile acid hydrophobicity. Furthermore, LKO and TKO decreased hepatic uptake and biliary secretion of high-density lipoprotein (HDL)-derived 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3ß-ol (NBD-cholesterol), while DKO alone had no effect. Finally, LKO and, to a lesser extent, DKO decreased most indexes contributing to cholesterol solubility in biliary bile. These results suggest different, but complementary, roles for L-FABP and SCP-2/SCP-x in biliary bile acid and cholesterol formation. L-FABP appears to function more in hepatic retention of bile acids as well as hepatic uptake and biliary secretion of HDL-cholesterol. Conversely, SCP-2/SCP-x may function more in formation and biliary secretion of bile acid, with less impact on hepatic uptake or biliary secretion of HDL-cholesterol.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Bilis/metabolismo , Proteínas Portadoras/fisiología , HDL-Colesterol/metabolismo , Proteínas de Unión a Ácidos Grasos/fisiología , Animales , Proteínas Portadoras/genética , Colesterol/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfolípidos/metabolismoRESUMEN
Although human liver fatty acid-binding protein (FABP1) T94A variant has been associated with nonalcoholic fatty liver disease and reduced ability of fenofibrate to lower serum triglycerides (TG) to target levels, molecular events leading to this phenotype are poorly understood. Cultured primary hepatocytes from female human subjects expressing the FABP1 T94A variant exhibited increased neutral lipid (TG, cholesteryl ester) accumulation associated with (1) upregulation of total FABP1, a key protein stimulating mitochondrial glycerol-3-phosphate acyltransferase (GPAM), the rate-limiting enzyme in lipogenesis; (2) increased mRNA expression of key enzymes in lipogenesis (GPAM, LPIN2) in heterozygotes; (3) decreased mRNA expression of microsomal triglyceride transfer protein; (4) increased secretion of ApoB100 but not TG; (5) decreased long-chain fatty acid (LCFA) ß-oxidation. TG accumulation was not due to any increase in LCFA uptake, de novo lipogenesis, or the alternate monoacylglycerol O-acyltransferase pathway in lipogenesis. Despite increased expression of total FABP1 mRNA and protein, fenofibrate-mediated FABP1 redistribution to nuclei and ligand-induced peroxisome proliferator-activated receptor (PPAR-α) transcription of LCFA ß-oxidative enzymes (carnitine palmitoyltransferase 1A, carnitine palmitoyltransferase 2, and acyl-coenzyme A oxidase 1, palmitoyl) were attenuated in FABP1 T94A hepatocytes. Although the phenotype of FABP1 T94A variant human hepatocytes exhibits some similarities to that of FABP1-null or PPAR-α-null hepatocytes and mice, expression of FABP1 T94A variant did not abolish or reduce ligand binding. Thus the FABP1 T94A variant represents an altered/reduced function mutation resulting in TG accumulation.
Asunto(s)
Proteínas de Unión a Ácidos Grasos/metabolismo , Ácidos Grasos/metabolismo , Hepatocitos/metabolismo , PPAR alfa/metabolismo , Apolipoproteína B-100/metabolismo , Células Cultivadas , Ésteres del Colesterol/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Femenino , Fenofibrato/farmacología , Regulación Enzimológica de la Expresión Génica , Variación Genética , Hepatocitos/efectos de los fármacos , Heterocigoto , Homocigoto , Humanos , Hipolipemiantes/farmacología , Lipogénesis/genética , Persona de Mediana Edad , Oxidación-Reducción , PPAR alfa/agonistas , Fenotipo , Unión Proteica , Transporte de Proteínas , ARN Mensajero/metabolismo , Factores de Tiempo , Transcripción Genética , Triglicéridos/metabolismoRESUMEN
Liver fatty acid binding protein (L-FABP) is the major soluble protein that binds very-long-chain n-3 polyunsaturated fatty acids (n-3 PUFAs) in hepatocytes. However, nothing is known about L-FABP's role in n-3 PUFA-mediated peroxisome proliferator activated receptor-α (PPARα) transcription of proteins involved in long-chain fatty acid (LCFA) ß-oxidation. This issue was addressed in cultured primary hepatocytes from wild-type, L-FABP-null, and PPARα-null mice with these major findings: 1) PUFA-mediated increase in the expression of PPARα-regulated LCFA ß-oxidative enzymes, LCFA/LCFA-CoA binding proteins (L-FABP, ACBP), and PPARα itself was L-FABP dependent; 2) PPARα transcription, robustly potentiated by high glucose but not maltose, a sugar not taken up, correlated with higher protein levels of these LCFA ß-oxidative enzymes and with increased LCFA ß-oxidation; and 3) high glucose altered the potency of n-3 relative to n-6 PUFA. This was not due to a direct effect of glucose on PPARα transcriptional activity nor indirectly through de novo fatty acid synthesis from glucose. Synergism was also not due to glucose impacting other signaling pathways, since it was observed only in hepatocytes expressing both L-FABP and PPARα. Ablation of L-FABP or PPARα as well as treatment with MK886 (PPARα inhibitor) abolished/reduced PUFA-mediated PPARα transcription of these genes, especially at high glucose. Finally, the PUFA-enhanced L-FABP distribution into nuclei with high glucose augmentation of the L-FABP/PPARα interaction reveals not only the importance of L-FABP for PUFA induction of PPARα target genes in fatty acid ß-oxidation but also the significance of a high glucose enhancement effect in diabetes.
Asunto(s)
Proteínas de Unión a Ácidos Grasos/farmacología , Ácidos Grasos Insaturados/farmacología , Glucosa/farmacología , PPAR alfa/biosíntesis , Acil-CoA Oxidasa/metabolismo , Animales , Western Blotting , Carnitina O-Palmitoiltransferasa/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Ácidos Grasos/biosíntesis , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Maltosa/farmacología , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Concentración Osmolar , PPAR alfa/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Ácidos Esteáricos/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Despite increasing awareness of the health risks associated with excess lipid storage in cells and tissues, knowledge of events governing lipid exchange at the surface of lipid droplets remains unclear. To address this issue, fluorescence resonance energy transfer (FRET) was performed to examine live cell interactions of Plin2 with lipids involved in maintaining lipid droplet structure and function. FRET efficiencies (E) between CFP-labeled Plin2 and fluorescently labeled phosphatidylcholine, sphingomyelin, stearic acid, and cholesterol were quantitated on a pixel-by-pixel basis to generate FRET image maps that specified areas with high E (>60%) in lipid droplets. The mean E and the distance R between the probes indicated a high yield of energy transfer and demonstrated molecular distances on the order of 44-57 Å, in keeping with direct molecular contact. In contrast, FRET between CFP-Plin2 and Nile red was not detected, indicating that the CFP-Plin2/Nile red interaction was beyond FRET proximity (>100 Å). An examination of the effect of Plin2 on cellular metabolism revealed that triacylglycerol, fatty acid, and cholesteryl ester content increased while diacylglycerol remained constant in CFP-Plin2-overexpressing cells. Total phospholipids also increased, reflecting increased phosphatidylcholine and sphingomyelin. Consistent with these results, expression levels of enzymes involved in triacylglycerol, cholesteryl ester, and phospholipid synthesis were significantly upregulated in CFP-Plin2-expressing cells while those associated with lipolysis either decreased or were unaffected. Taken together, these data show for the first time that Plin2 interacts directly with lipids on the surface of lipid droplets and influences levels of key enzymes and lipids involved in maintaining lipid droplet structure and function.
Asunto(s)
Fibroblastos/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Metabolismo de los Lípidos/fisiología , Proteínas de la Membrana/metabolismo , Animales , Células Cultivadas , Humanos , Ratones , Perilipina-2 , Unión Proteica/fisiología , Relación Estructura-Actividad , Propiedades de SuperficieRESUMEN
Although lipid-rich microdomains of hepatocyte plasma membranes serve as the major scaffolding regions for cholesterol transport proteins important in cholesterol disposition, little is known regarding intracellular factors regulating cholesterol distribution therein. On the basis of its ability to bind cholesterol and alter hepatic cholesterol accumulation, the cytosolic liver type FA binding protein (L-FABP) was hypothesized to be a candidate protein regulating these microdomains. Compared with wild-type hepatocyte plasma membranes, L-FABP gene ablation significantly increased the proportion of cholesterol-rich microdomains. Lack of L-FABP selectively increased cholesterol, phospholipid (especially phosphatidylcholine), and branched-chain FA accumulation in the cholesterol-rich microdomains. These cholesterol-rich microdomains are important, owing to enrichment therein of significant amounts of key transport proteins involved in uptake of cholesterol [SR-B1, ABCA-1, P-glycoprotein (P-gp), sterol carrier binding protein (SCP-2)], FA transport protein (FATP), and glucose transporters 1 and 2 (GLUT1, GLUT2) insulin receptor. L-FABP gene ablation enhanced the concentration of SCP-2, SR-B1, FATP4, and GLUT1 in the cholesterol-poor microdomains, with functional implications in HDL-mediated uptake and efflux of cholesterol. Thus L-FABP gene ablation significantly impacted the proportion of cholesterol-rich versus -poor microdomains in the hepatocyte plasma membrane and altered the distribution of lipids and proteins involved in cholesterol uptake therein.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Microdominios de Membrana/metabolismo , Animales , Western Blotting , Membrana Celular/genética , Células Cultivadas , Colesterol/genética , Colesterol/metabolismo , Proteínas de Transporte de Ácidos Grasos/genética , Proteínas de Transporte de Ácidos Grasos/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Microdominios de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Microscopía Confocal , Fosfolípidos/química , Fosfolípidos/metabolismoRESUMEN
The liver expresses high levels of two proteins with high affinity for long-chain fatty acids (LCFAs): liver fatty acid binding protein (L-FABP) and sterol carrier protein-2 (SCP-2). Real-time confocal microscopy of cultured primary hepatocytes from gene-ablated (L-FABP, SCP-2/SCP-x, and L-FABP/SCP-2/SCP-x null) mice showed that the loss of L-FABP reduced cellular uptake of 12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazo)-aminostearic acid (a fluorescent-saturated LCFA analog) by â¼50%. Importantly, nuclear targeting of the LCFA was enhanced when L-FABP was upregulated (SCP-2/SCP-x null) but was significantly reduced when L-FABP was ablated (L-FABP null), thus impacting LCFA nuclear targeting. These effects were not associated with a net decrease in expression of key membrane proteins involved in LCFA or glucose transport. Since hepatic LCFA uptake and metabolism are closely linked to glucose uptake, the effect of glucose on L-FABP-mediated LCFA uptake and nuclear targeting was examined. Increasing concentrations of glucose decreased cellular LCFA uptake and even more extensively decreased LCFA nuclear targeting. Loss of L-FABP exacerbated the decrease in LCFA nuclear targeting, while loss of SCP-2 reduced the glucose effect, resulting in enhanced LCFA nuclear targeting compared with control. Simply, ablation of L-FABP decreases LCFA uptake and even more extensively decreases its nuclear targeting.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Hepatocitos/fisiología , Ácidos Esteáricos/metabolismo , Animales , Transporte Biológico/fisiología , Western Blotting , Células Cultivadas , Glucosa/metabolismo , Metabolismo de los Lípidos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Regulación hacia Arriba/fisiologíaRESUMEN
A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3ß-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in <1 min and initial rate and maximal uptake of HDL free cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2/SCP-x/L-FABP null mice and hepatocytes. Taken together, these results suggest that L-FABP, particularly in the absence of SCP-2, plays a significant role in HDL-mediated cholesterol uptake in cultured primary hepatocytes.
Asunto(s)
Proteínas Portadoras/metabolismo , HDL-Colesterol/metabolismo , Colesterol/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Hepatocitos/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/farmacología , Animales , Western Blotting , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Membrana Celular/química , Membrana Celular/metabolismo , Separación Celular , Células Cultivadas , Colesterol/análogos & derivados , Colesterol/farmacología , Proteínas de Unión a Ácidos Grasos/biosíntesis , Proteínas de Unión a Ácidos Grasos/genética , Inmunohistoquímica , Lipoproteínas/biosíntesis , Lipoproteínas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía InmunoelectrónicaRESUMEN
The significance of lipid droplets (LD) in lipid metabolism, cell signaling, and membrane trafficking is increasingly recognized, yet the role of the LD phospholipid monolayer in LD protein targeting and function remains unknown. To begin to address this issue, two populations of LD were isolated by ConA sepharose affinity chromatography: 1) functionally active LD enriched in perilipin, caveolin-1, and several lipolytic proteins, including ATGL and HSL; and 2) LD enriched in ADRP and TIP47 that contained little to no lipase activity. Coimmunoprecipitation experiments confirmed the close association of caveolin and perilipin and lack of interaction between caveolin and ADRP, in keeping with the separation observed with the ConA procedure. The phospholipid monolayer structure was evaluated to reveal that the perilipin-enriched LD exhibited increased rigidity (less fluidity), as shown by increased cholesterol/phospholipid, Sat/Unsat, and Sat/MUFA ratios. These results were confirmed by DPH-TMA, NBD-cholesterol, and NBD-sphingomyelin fluorescence polarization studies. By structure and organization, the perilipin-enriched LD most closely resembled the adipocyte PM. In contrast, the ADRP/TIP47-enriched LD contained a more fluid monolayer membrane, reflecting decreased polarizations and lipid order based on phospholipid fatty acid analysis. Taken together, results indicate that perilipin and associated lipolytic enzymes target areas in the phospholipid monolayer that are highly organized and rigid, similar in structure to localized areas of the PM where cholesterol and fatty acid uptake and efflux occur.
Asunto(s)
Proteínas Portadoras/metabolismo , Vesículas Citoplasmáticas/metabolismo , Fluidez de la Membrana , Fosfoproteínas/metabolismo , Adipocitos/química , Adipocitos/metabolismo , Animales , Membrana Celular/química , Membrana Celular/metabolismo , Estructuras de la Membrana Celular/química , Estructuras de la Membrana Celular/metabolismo , Células Cultivadas , Cromatografía de Afinidad , Vesículas Citoplasmáticas/química , Metabolismo de los Lípidos/fisiología , Masculino , Fluidez de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Perilipina-1 , Fosfolípidos/química , Fosfolípidos/metabolismo , Fracciones Subcelulares/química , Fracciones Subcelulares/metabolismoRESUMEN
BACKGROUND: Rotavirus NSP4 localizes to multiple intracellular sites and is multifunctional, contributing to RV morphogenesis, replication and pathogenesis. One function of NSP4 is the induction of early secretory diarrhea by binding surface receptors to initiate signaling events. The aims of this study were to determine the transport kinetics of NSP4 to the exofacial plasma membrane (PM), the subsequent release from intact infected cells, and rebinding to naïve and/or neighboring cells in two cell types. METHODS: Transport kinetics was evaluated using surface-specific biotinylation/streptavidin pull-downs and exofacial exposure of NSP4 was confirmed by antibody binding to intact cells, and fluorescent resonant energy transfer. Transfected cells similarly were monitored to discern NSP4 movement in the absence of infection or other viral proteins. Endoglycosidase H digestions, preparation of CY3- or CY5- labeled F(ab)2 fragments, confocal imaging, and determination of preferential polarized transport employed standard laboratory techniques. Mock-infected, mock-biotinylated and non-specific antibodies served as controls. RESULTS: Only full-length (FL), endoglycosidase-sensitive NSP4 was detected on the exofacial surface of two cell types, whereas the corresponding cell lysates showed multiple glycosylated forms. The C-terminus of FL NSP4 was detected on exofacial-membrane surfaces at different times in different cell types prior to its release into culture media. Transport to the PM was rapid and distinct yet FL NSP4 was secreted from both cell types at a time similar to the release of virus. NSP4-containing, clarified media from both cells bound surface molecules of naïve cells, and imaging showed secreted NSP4 from one or more infected cells bound neighboring cell membranes in culture. Preferential sorting to apical or basolateral membranes also was distinct in different polarized cells. CONCLUSIONS: The intracellular transport of NSP4 to the PM, translocation across the PM, exposure of the C-terminus on the cell surface and subsequent secretion occurs via an unusual, complex and likely cell-dependent process. The exofacial exposure of the C-terminus poses several questions and suggests an atypical mechanism by which NSP4 traverses the PM and interacts with membrane lipids. Mechanistic details of the unconventional trafficking of NSP4, interactions with host-cell specific molecules and subsequent release require additional study.
Asunto(s)
Membrana Celular/metabolismo , Glicoproteínas/metabolismo , Rotavirus/patogenicidad , Toxinas Biológicas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Línea Celular , Humanos , Cinética , Unión Proteica , Transporte de Proteínas , Coloración y Etiquetado/métodosRESUMEN
Although HDL-mediated cholesterol transport to the liver is well studied, cholesterol efflux from hepatocytes back to HDL is less well understood. Real-time imaging of efflux of 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino)-23,24-bisnor-5-cholen-3beta-ol (NBD-cholesterol), which is poorly esterified, and [(3)H]cholesterol, which is extensively esterified, from cultured primary hepatocytes of wild-type and sterol carrier protein-2 (SCP-2) gene-ablated mice showed that 1) NBD-cholesterol efflux was affected by the type of lipoprotein acceptor, i.e., HDL3 over HDL2; 2) NBD-cholesterol efflux was rapid (detected in 1-2 min) and resolved into fast [half time (t((1/2))) = 2.4 min, 6% of total] and slow (t((1/2)) = 26.5 min, 94% of total) pools, consistent with protein- and vesicle-mediated cholesterol transfer, respectively; 3) SCP-2 gene ablation increased efflux of NBD-cholesterol, as well as [(3)H]cholesterol, albeit less so due to competition by esterification of [(3)H]cholesterol, but not NBD-cholesterol; and 4) SCP-2 gene ablation increased initial rate (2.3-fold) and size (9.7-fold) of rapid effluxing sterol, suggesting an increased contribution of molecular cholesterol transfer. In addition, colocalization, double-immunolabeling fluorescence resonance energy transfer, and electron microscopy, as well as cross-linking coimmunoprecipitation, indicated that SCP-2 directly interacted with the HDL receptor, scavenger receptor class B type 1 (SRB1), in hepatocytes. Other membrane proteins in cholesterol efflux [SRB1 and ATP-binding cassettes (ABC) A-1, ABCG-1, ABCG-5, and ABCG-8] and several soluble/vesicle-associated proteins facilitating intracellular cholesterol trafficking (StARDs, NPCs, ORPs) were not upregulated. However, loss of SCP-2 elicited twofold upregulation of liver fatty acid-binding protein (L-FABP), a protein with lower affinity for cholesterol but higher cytosolic concentration than SCP-2. Ablation of SCP-2 and L-FABP decreased HDL-mediated NBD-cholesterol efflux. These results indicate that SCP-2 expression plays a significant role in HDL-mediated cholesterol efflux by regulating the size of rapid vs. slow cholesterol efflux pools and/or eliciting concomitant upregulation of L-FABP in cultured primary hepatocytes.
Asunto(s)
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Proteínas Portadoras/metabolismo , Colesterol/análogos & derivados , Hepatocitos/metabolismo , Lipoproteínas HDL3/metabolismo , 4-Cloro-7-nitrobenzofurazano/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Transporte Biológico , Proteínas Portadoras/genética , Técnicas de Cultivo de Célula , Células Cultivadas , Colesterol/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Técnicas de Inactivación de Genes , Inmunoprecipitación , Cinética , Lipoproteínas HDL2/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía Electrónica , Fosfoproteínas/metabolismo , Unión Proteica , Receptores Depuradores de Clase B/metabolismo , Vesículas Transportadoras/metabolismoRESUMEN
Although studies with liver type fatty acid binding protein (L-FABP) gene ablated mice demonstrate a physiological role for L-FABP in hepatic fatty acid metabolism, little is known about the mechanisms whereby L-FABP elicits these effects. Studies indicate that L-FABP may function to shuttle lipids to the nucleus, thereby increasing the availability of ligands of nuclear receptors, such as peroxisome proliferator-activated receptor-alpha (PPARalpha). The data herein suggest that such mechanisms involve direct interaction of L-FABP with PPARalpha. L-FABP was shown to directly interact with PPARalpha in vitro through co-immunoprecipitation (co-IP) of pure proteins, altered circular dichroic (CD) spectra, and altered fluorescence spectra. In vitro fluorescence resonance energy transfer (FRET) between Cy3-labeled PPARalpha and Cy5-labeled L-FABP proteins showed that these proteins bound with high affinity (Kd approximately 156 nM) and in close proximity (intermolecular distance of 52A). This interaction was further substantiated by co-IP of both proteins from liver homogenates of wild-type mice. Moreover, double immunogold electron microscopy and FRET confocal microscopy of cultured primary hepatocytes showed that L-FABP was in close proximity to PPARalpha (intermolecular distance 40-49A) in vivo. Taken together, these studies were consistent with L-FABP regulating PPARalpha transcriptional activity in hepatocytes through direct interaction with PPARalpha. Our in vitro and imaging experiments demonstrate high affinity, structural molecular interaction of L-FABP with PPARalpha and suggest a functional role for L-FABP interaction with PPARalpha in long chain fatty acid (LCFA) metabolism.
Asunto(s)
Proteínas de Unión a Ácidos Grasos/metabolismo , Hepatocitos/metabolismo , PPAR alfa/metabolismo , Animales , Compartimento Celular , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas de Unión a Ácidos Grasos/química , Ácidos Grasos/metabolismo , Hepatocitos/ultraestructura , Ligandos , Masculino , Ratones , Ratones Noqueados , PPAR alfa/química , Ácido Palmítico/metabolismo , Unión Proteica , Conformación Proteica , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismoRESUMEN
Liver fatty acid binding protein (L-FABP) is the major fatty acid binding/"chaperone" protein in hepatic cytosol. Although fatty acids can be derived from the breakdown of dietary fat and glucose, relatively little is known regarding the impact of L-FABP on phenotype in the context of high dietary glucose. Potential impact was examined in wild-type (WT) and Lfabp gene ablated (LKO) female mice fed either a control or pair-fed high glucose diet (HGD). WT mice fed HGD alone exhibited decreased whole body weight gain and weight gain/kcal food consumed-both as reduced lean tissue mass (LTM) and fat tissue mass (FTM). Conversely, LKO alone increased weight gain, lean tissue mass, and fat tissue mass while decreasing serum ß-hydroxybutyrate (indicative of hepatic fatty acid oxidation)-regardless of diet. Both LKO alone and HGD alone significantly altered the serum lipoprotein profile and increased triacylglycerol (TG), but in HGD mice the LKO did not further exacerbate serum TG content. HGD had little effect on hepatic lipid composition in WT mice, but prevented the LKO-induced selective increase in hepatic phospholipid, free-cholesterol and cholesteryl-ester. Taken together, these findings suggest that high glucose diet diminished the effects of LKO on the whole body and lipid phenotype of these mice.
Asunto(s)
Proteínas de Unión a Ácidos Grasos/deficiencia , Glucosa/farmacología , Metabolismo de los Lípidos , Animales , Colesterol/metabolismo , Dieta , Proteínas de Unión a Ácidos Grasos/genética , Femenino , Hígado/metabolismo , Ratones , Fosfolípidos/metabolismo , Triglicéridos/metabolismo , Aumento de PesoRESUMEN
Sterol carrier protein-2 (SCP-2) was independently discovered as a soluble protein that binds and transfers cholesterol as well as phospholipids (nonspecific lipid transfer protein, nsLTP) in vitro. Physiological functions of this protein are only now beginning to be resolved. The gene encoding SCP-2 also encodes sterol carrier protein-x (SCP-x) arising from an alternate transcription site. In vitro and in vivo SCP-x serves as a peroxisomal 3-ketoacyl-CoA thiolase in oxidation of branched-chain lipids (cholesterol to form bile acids; branched-chain fatty acid for detoxification). While peroxisomal SCP-2 facilitates branched-chain lipid oxidation, the role(s) of extraperoxisomal (up to 50% of total) are less clear. Studies using transfected fibroblasts overexpressing SCP-2 and hepatocytes from SCP-2/SCP-x gene-ablated mice reveal that SCP-2 selectively remodels the lipid composition, structure, and function of lipid rafts/caveolae. Studies of purified SCP-2 and in cells show that SCP-2 has high affinity for and selectively transfers many lipid species involved in intracellular signaling: fatty acids, fatty acyl CoAs, lysophosphatidic acid, phosphatidylinositols, and sphingolipids (sphingomyelin, ceramide, mono-di-and multi-hexosylceramides, gangliosides). SCP-2 selectively redistributes these signaling lipids between lipid rafts/caveolae and intracellular sites. These findings suggest SCP-2 serves not only in cholesterol and phospholipid transfer, but also in regulating multiple lipid signaling pathways in lipid raft/caveolae microdomains of the plasma membrane.
Asunto(s)
Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Metabolismo de los Lípidos , Microdominios de Membrana/metabolismo , Transducción de Señal , Animales , Transporte Biológico , Proteínas Portadoras/genética , Línea Celular , Colesterol/metabolismo , Ácidos Grasos/metabolismo , Humanos , Lípidos de la Membrana/metabolismo , Microdominios de Membrana/química , Ratones , Modelos Biológicos , Esteroles/metabolismo , Relación Estructura-ActividadRESUMEN
Dehydroergosterol [ergosta-5,7,9(11),22-tetraen-3beta-ol] is a naturally-occurring, fluorescent sterol utilized extensively to probe membrane cholesterol distribution, cholesterol-protein interactions, and intracellular cholesterol transport both in vitro and in vivo. In aqueous solutions, the low solubility of dehydroergosterol results in the formation of monohydrate crystals similar to cholesterol. Low temperature X-ray diffraction analysis reveals that dehydroergosterol monohydrate crystallizes in the space group P2(1) with four molecules in the unit cell and monoclinic crystal parameters a = 9.975(1) A, b = 7.4731(9) A, c = 34.054(4) A, and beta = 92.970(2) degrees somewhat similar to ergosterol monohydrate. The molecular arrangement is in a slightly closer packed bilayer structure resembling cholesterol monohydrate. Since dehydroergosterol fluorescence emission undergoes a quantum yield enhancement and red-shift of its maximum wavelength when crystallized, formation or disruption of microcrystals was monitored with high sensitivity using cuvette-based spectroscopy and multi-photon laser scanning imaging microscopy. This manuscript reports on the dynamical effect of sterol carrier protein-2 (SCP-2) interacting between aqueous dispersions of dehydroergosterol monohydrate microcrystal donors and acceptors consisting not only of model membranes but also vesicles derived from plasma membranes isolated by biochemical fractionation and affinity purification from Madin-Darby canine kidney cells. Furthermore, this study provides real-time measurements of the effect of increased SCP-2 levels on the rate of disappearance of dehydroergosterol microcrystals in living cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Ergosterol/análogos & derivados , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Cristalización , Ergosterol/química , Humanos , Ratones , Estructura Molecular , Esteroles/metabolismo , Difracción de Rayos XRESUMEN
Loss of liver fatty acid binding protein (L-FABP) decreases long chain fatty acid uptake and oxidation in primary hepatocytes and in vivo. On this basis, L-FABP gene ablation would potentiate high-fat diet-induced weight gain and weight gain/energy intake. While this was indeed the case when L-FABP null (-/-) mice on the C57BL/6NCr background were pair-fed a high-fat diet, whether this would also be observed under high-fat diet fed ad libitum was not known. Therefore, this possibility was examined in female L-FABP (-/-) mice on the same background. L-FABP (-/-) mice consumed equal amounts of defined high-fat or isocaloric control diets fed ad libitum. However, on the ad libitum-fed high-fat diet the L-FABP (-/-) mice exhibited: (1) decreased hepatic long chain fatty acid (LCFA) ß-oxidation as indicated by lower serum ß-hydroxybutyrate level; (2) decreased hepatic protein levels of key enzymes mitochondrial (rate limiting carnitine palmitoyl acyltransferase A1, CPT1A; HMG-CoA synthase) and peroxisomal (acyl CoA oxidase 1, ACOX1) LCFA ß-oxidation; (3) increased fat tissue mass (FTM) and FTM/energy intake to the greatest extent; and (4) exacerbated body weight gain, weight gain/energy intake, liver weight, and liver weight/body weight to the greatest extent. Taken together, these findings showed that L-FABP gene-ablation exacerbated diet-induced weight gain and fat tissue mass gain in mice fed high-fat diet ad libitum--consistent with the known biochemistry and cell biology of L-FABP.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Proteínas de Unión a Ácidos Grasos/genética , Ácidos Grasos/metabolismo , Eliminación de Gen , Hígado/metabolismo , Aumento de Peso , Ácido 3-Hidroxibutírico/sangre , Ácido 3-Hidroxibutírico/metabolismo , Acil-CoA Oxidasa/metabolismo , Tejido Adiposo/anatomía & histología , Tejido Adiposo/metabolismo , Animales , Ingestión de Alimentos , Proteínas de Transporte de Ácidos Grasos/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Femenino , Hepatocitos/metabolismo , Hidroximetilglutaril-CoA Sintasa/metabolismo , Hígado/anatomía & histología , Hígado/enzimología , Ratones , Ratones Endogámicos C57BL , Tamaño de los Órganos , Oxidación-ReducciónRESUMEN
During the last several years, intracellular lipid droplets have become the focus of intense study. No longer an inert bystander, the lipid droplet is now known as a dynamic organelle contributing lipids to many cellular events. However, while the dynamics of cholesterol efflux from both the plasma membrane and lipid droplets have been studied, less is known regarding the efflux of sphingomyelin from these membranes. In order to address this issue, sphingomyelin efflux kinetics and binding affinities from different intracellular pools were examined. When compared to the plasma membrane, lipid droplets had a smaller exchangeable sphingomyelin efflux pool and the time required to efflux that pool was significantly shorter. Fluorescence binding assays revealed that proteins in the plasma membrane and lipid droplet pool bound sphingomyelin with high affinity. Further characterization identified adipose differentiation-related protein (ADRP) as one of the sphingomyelin binding proteins in the lipid droplet fraction and revealed that ADRP demonstrated saturable binding to 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-hexanoyl)sphingosyl-phosphocholine (NBD-sphingomyelin) and also 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-phosphatidylcholine) with binding affinities in the nanomolar range. Taken together, these results suggest that lipid droplet associated proteins such as ADRP may play a significant role in regulating the intracellular distribution of phospholipids and lipids in general. Overall, insights from the present work suggest new and important roles for lipid droplets and ADRP in phospholipid metabolism.