RESUMEN
The objective of this study was to measure the effects of a Lactobacillus fermentation product (LBFP) on fecal characteristics and microbiota, blood biomarkers, immune function, and serum oxidative stress markers of adult dogs. Thirty adult beagle dogs [23 M, 7 F; mean age = 8.47 ± 2.65 yr old; mean BW = 15.43 ± 4.17 kg] were used in a completely randomized design study. All dogs were fed a basal diet to maintain BW for 5 wk, followed by baseline blood and fecal sample collections. Dogs remained on the same diet, but then were randomly assigned to a placebo (dextrose) or LBFP supplement (Limosilactobacillus fermentum and Lactobacillus delbrueckii). Both treatments were dosed at 4 mg/kg BW via gelatin capsule for 5 wk (n = 15/treatment). Fecal and blood samples were collected at that time. Change from baseline data were analyzed using the Mixed Models procedure of SAS 9.4, with P < 0.05 being significant and P < 0.10 being trends. Most circulating metabolites and immunoglobulins (Ig) were unaltered by treatment, but LBFP-supplemented dogs had lower changes in serum corticosteroid isoenzyme of alkaline phosphatase (P < 0.05), alanine aminotransferase (P < 0.10), and IgM (P < 0.10) than controls. The change in fecal scores tended to be lower (P = 0.068) in LBFP-supplemented dogs than controls, signifying firmer feces in LBFP-supplemented dogs. Regarding the fecal microbiota, alpha diversity indicators tended to be higher (P = 0.087) in LBFP-supplemented dogs than controls. One fecal bacterial phylum (Actinobacteriota) was altered by treatments, with its relative abundance tending to have a greater (P < 0.10) increase in controls than LBFP-supplemented dogs. Fifteen bacterial genera were altered (P < 0.05 or P < 0.10) by treatments, including relative abundances of fecal Peptoclostridium, Sarcina, and Faecalitalea that had a greater (P < 0.05) increase in controls than LBFP-supplemented dogs. In contrast, relative abundances of fecal Faecalibaculum, Bifidobacterium, and uncultured Butyricicoccaceae had a greater (P ≤ 0.05) increase in LBFP-supplemented dogs than controls. After week 5, dogs underwent transport stress (45-min vehicle ride) to assess oxidative stress markers. The change in serum superoxide dismutase after transport had a greater (P < 0.0001) increase in LBFP-supplemented dogs than controls. Our data suggest that LBFP may provide benefits to dogs by stabilizing stool quality, beneficially shifting fecal microbiota, and protecting against oxidative damage when subjected to stress.
Our objective was to measure the effects of a Lactobacillus fermentation product (LBFP) on fecal characteristics and microbiota, immune function, and oxidative stress markers of dogs. Thirty adult dogs were used in a completely randomized design study. All dogs were fed a basal diet to maintain body weight for 5 wk and then randomly assigned to a placebo or LBFP supplement for five more weeks. Fecal and blood samples were collected after baseline and treatment phases. Change from baseline data were analyzed statistically. Most blood markers were unaltered by treatment, but LBFP-supplemented dogs had lower changes in liver enzymes and IgM than controls. Change in fecal scores tended to be lower in LBFP-supplemented dogs than controls, signifying firmer feces. Fecal bacterial alpha diversity tended to be higher in LBFP-supplemented dogs than controls. One fecal bacterial phylum and 15 bacterial genera were altered by treatments. After 5 wk, dogs underwent transport stress (45-min vehicle ride) to assess oxidative stress markers. The increase in serum superoxide dismutase after transport was greater in LBFP-supplemented dogs than controls. Our data suggest that LBFP may provide benefits to dogs by stabilizing stool quality, beneficially shifting fecal microbiota, and protecting against oxidative damage when undergoing stress.
Asunto(s)
Dieta , Lactobacillus , Perros , Animales , Fermentación , Heces/microbiología , Dieta/veterinaria , Inmunidad , Alimentación Animal/análisisRESUMEN
BACKGROUND: The prevalence of peanut allergies is increasing. Peanuts and many other allergen sources contain significant amounts of triglycerides, which affect absorption of antigens but have unknown effects on sensitization and anaphylaxis. We recently reported that dietary medium-chain triglycerides (MCTs), which bypass mesenteric lymph and directly enter portal blood, reduce intestinal antigen absorption into blood compared with long-chain triglycerides (LCTs), which stimulate mesenteric lymph flow and are absorbed in chylomicrons through mesenteric lymph. OBJECTIVE: We sought to test how dietary MCTs affect food allergy. METHODS: C3H/HeJ mice were fed peanut butter protein in MCT, LCT (peanut oil), or LCT plus an inhibitor of chylomicron formation (Pluronic L81). Peanut-specific antibodies in plasma, responses of the mice to antigen challenges, and intestinal epithelial cytokine expression were subsequently measured. RESULTS: MCT suppressed antigen absorption into blood but stimulated absorption into Peyer patches. A single gavage of peanut protein with MCT, as well as prolonged feeding in MCT-based diets, caused spontaneous allergic sensitization. MCT-sensitized mice experienced IgG-dependent anaphylaxis on systemic challenge and IgE-dependent anaphylaxis on oral challenge. MCT feeding stimulated jejunal-epithelial thymic stromal lymphopoietin, Il25, and Il33 expression compared with that seen after LCT feeding and promoted T(H)2 cytokine responses in splenocytes. Moreover, oral challenges of sensitized mice with antigen in MCT significantly aggravated anaphylaxis compared with challenges with the LCT. Importantly, the effects of MCTs could be mimicked by adding Pluronic L81 to LCTs, and in vitro assays indicated that chylomicrons prevent basophil activation. CONCLUSION: Dietary MCTs promote allergic sensitization and anaphylaxis by affecting antigen absorption and availability and by stimulating T(H)2 responses.
Asunto(s)
Anafilaxia/inmunología , Arachis/inmunología , Hipersensibilidad al Cacahuete/inmunología , Triglicéridos/inmunología , Alérgenos/inmunología , Anafilaxia/metabolismo , Animales , Anticuerpos/inmunología , Antígenos/inmunología , Arachis/química , Basófilos/inmunología , Basófilos/metabolismo , Quilomicrones/inmunología , Citocinas/inmunología , Dieta/métodos , Femenino , Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Interleucinas/inmunología , Absorción Intestinal/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Yeyuno/inmunología , Yeyuno/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratas , Ratas Sprague-Dawley , Células Th2/inmunología , Células Th2/metabolismo , Triglicéridos/administración & dosificación , Triglicéridos/metabolismoRESUMEN
BACKGROUND: Serum Amyloid A (SAA) is a major acute phase protein of unknown function. SAA is mostly expressed in the liver, but also in other tissues including the intestinal epithelium. SAA reportedly has anti-bacterial effects, and because inflammatory bowel diseases (IBD) result from a breakdown in homeostatic interactions between intestinal epithelia and bacteria, we hypothesized that SAA is protective during experimental colitis. METHODS: Intestinal SAA expression was measured in mouse and human samples. Dextran sodium sulfate (DSS) colitis was induced in SAA 1/2 double knockout (DKO) mice and in wildtype controls. Anti-bacterial effects of SAA1/2 were tested in intestinal epithelial cell lines transduced with adenoviral vectors encoding the CE/J SAA isoform or control vectors prior to exposure to live Escherichia coli. RESULTS: Significant levels of SAA1/SAA2 RNA and SAA protein were detected by in situ hybridization and immunohistochemistry in mouse colonic epithelium. SAA3 expression was weaker, but similarly distributed. SAA1/2 RNA was present in the ileum and colon of conventional mice and in the colon of germfree mice. Expression of SAA3 was strongly regulated by bacterial lipopolysaccharides in cultured epithelial cell lines, whereas SAA1/2 expression was constitutive and not LPS inducible. Overexpression of SAA1/2 in cultured epithelial cell lines reduced the viability of co-cultured E. coli. This might partially explain the observed increase in susceptibility of DKO mice to DSS colitis. SAA1/2 expression was increased in colon samples obtained from Crohn's Disease patients compared to controls. CONCLUSIONS: Intestinal epithelial SAA displays bactericidal properties in vitro and could play a protective role in experimental mouse colitis. Altered expression of SAA in intestinal biopsies from Crohn's Disease patients suggests that SAA is involved in the disease process..
Asunto(s)
Bacterias/crecimiento & desarrollo , Colitis/genética , ADN/genética , Regulación de la Expresión Génica , Proteína Amiloide A Sérica/genética , Animales , Bacterias/efectos de los fármacos , Biopsia , Línea Celular , Colitis/microbiología , Colitis/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Immunoblotting , Hibridación in Situ , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Proteína Amiloide A Sérica/biosíntesisRESUMEN
Little is known about the release and intercellular transport of Wnt proteins from mammalian cells. Lipoproteins may act as carriers for the intercellular movement and gradient formation of the lipid-linked morphogens Wingless and Hedgehog in Drosophila. To investigate whether such a mechanism can occur in mammals, we have studied Wnt release in cultured mammalian cells. Wnt3a associated with lipoproteins in the culture medium and not with extracellular vesicles or exosomes. Although Wnt3a was associated with both high-density lipoproteins (HDL) and low-density lipoproteins, only HDL allowed Wnt3a release from mouse fibroblasts. Remarkably, Wnt3a lacking its palmitate moiety was released in a lipoprotein-independent manner, demonstrating the dual role of palmitoylation in membrane and lipoprotein binding. We additionally found that Wnt3a can be released from enterocyte cell lines on endogenously expressed lipoproteins. We further discuss the physiological implications of our findings.
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Lipoproteínas/metabolismo , Proteínas Wnt/metabolismo , Animales , Línea Celular , Cricetinae , Proteínas de Transporte de Ácidos Grasos/metabolismo , Proteínas Wnt/genética , Proteína Wnt3 , Proteína Wnt3ARESUMEN
BACKGROUND: Osteopontin (OPN), a secreted glycoprotein that promotes TH1 immune responses, is involved in several inflammatory conditions. Recently, OPN plasma levels have been demonstrated to be elevated in patients with Crohn's disease. From this evidence, we investigated in the present study whether OPN deficiency protects mice against dextran sodium sulfate (DSS)-induced colitis. MATERIALS AND METHODS: Colitis was induced in OPN -/- mice and matched wild-type Black Swiss control mice by adding 3.5% DSS to their drinking water. Disease progression was evaluated for 10 days by measuring body weight, stool consistency, rectal bleeding, colon lengths, histology, and immunohistochemistry. Levels of the acute-phase protein serum amyloid A, O PN, the proinflammatory cytokines interleukin (IL)-6 and IL-12, and the anti-inflammatory cytokine IL-10 were measured in the serum and, in the case of IL-10 and IL-12, in supernatants from colonic explants at the end of treatment. RESULTS: After DSS treatment, OPN -/- mice exhibited significantly decreased disease activity compared with wild-type mice, as evidenced by reduced rectal bleeding, weight loss, and histological intestinal injury (P < 0.002). Furthermore, serum levels of serum amyloid A and IL-6 increased to a lesser extent (P < 0.001), which also was the case for the release of IL-12 by colonic explants (P < 0.01). The release of IL-10 by colonic explants, however, was increased (P < 0.01). Serum levels of IL-10 and IL-12 were not affected by DSS treatment in both wild-type and OPN-/- mice. Macrophage infiltration into inflamed colonic tissue also was markedly attenuated in DSS-treated OPN -/- mice compared with wild-type mice. CONCLUSIONS: This study shows that OPN deficiency significantly protected mice from colitis by attenuating the TH1 response and macrophage chemotaxis. OPN may represent a novel attractive target for pharmacological treatment of inflammatory bowel disease.
Asunto(s)
Colitis/prevención & control , Citocinas/metabolismo , Proteína Amiloide A Sérica/metabolismo , Sialoglicoproteínas/deficiencia , Administración Oral , Animales , Colitis/inducido químicamente , Colitis/metabolismo , Colon/patología , Citocinas/sangre , Sulfato de Dextran , Ratones , Ratones Transgénicos , OsteopontinaRESUMEN
OBJECTIVE: The high-density lipoprotein (HDL) receptor scavenger receptor Class B type I (SR-BI) plays a key role in mediating the final step of reverse cholesterol transport. This study examined the possible regulation of hepatic SR-BI by phosphatidylinositol-3-kinase (PI3K), a well known regulator of endocytosis and membrane protein trafficking. METHODS AND RESULTS: SR-BI-dependent HDL selective cholesterol ester uptake in human HepG2 hepatoma cells was decreased (approximately 50%) by the PI3K inhibitors wortmannin and LY294002. Insulin increased selective uptake (approximately 30%), and this increase was blocked by PI3K inhibitors. Changes in SR-BI activity could be accounted for by pronounced changes in the subcellular localization and cell surface expression of SR-BI as determined by HDL cell surface binding, receptor biotinylation studies, and confocal fluorescence microscopy of HepG2 cells expressing green fluorescent protein-tagged SR-BI. Thus, under conditions of PI3K activation by insulin, and to a lesser extent by the SR-BI ligand HDL, cell surface expression of SR-BI was promoted, resulting in increased SR-BI-mediated HDL selective lipid uptake. CONCLUSIONS: Our data indicate that PI3K activation stimulates hepatic SR-BI function post-translationally by regulating the subcellular localization of SR-BI in a P13K-dependent manner. Decreased hepatocyte PI3K activity in insulin-resistant states, such as type 2 diabetes, obesity, or metabolic syndrome, may impair reverse cholesterol transport by reducing cell surface expression of SR-BI.
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Hepatocitos/metabolismo , Metabolismo de los Lípidos/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Receptores Depuradores de Clase B/metabolismo , Fracciones Subcelulares/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Humanos , Insulina/farmacología , Lipoproteínas HDL/farmacología , Receptores Depuradores de Clase B/clasificación , Distribución Tisular/efectos de los fármacos , Distribución Tisular/fisiologíaRESUMEN
Previous studies have suggested that HDL retroendocytosis may play a role in scavenger receptor class B type I (SR-BI)-dependent selective lipid uptake in a cell-specific manner. To investigate this possibility, we developed methods to quantitatively measure HDL uptake and resecretion in fibroblast (COS-7) and hepatocyte (HepG2) cells expressing exogenous SR-BI. Approximately 17% and 24% of HDL associated in an SR-BI-dependent manner with COS-7 and HepG2 cells, respectively, accumulates intracellularly after a 10 min incubation. To determine whether this intracellular HDL undergoes retroendocytosis, we developed a pulse-chase assay whereby internalized biotinylated (125)I-HDL(3) secreted from cells is quantitatively precipitated from cell supernatants using immobilized streptavidin. Our results show a rapid secretion of a portion of intracellular HDL from both cell types (representing 4-7% of the total cell-associated HDL) that is almost complete within 30 min (half-life approximately 10 min). In COS-7 cells, the calculated rate of HDL secretion ( approximately 0.5 ng HDL/mg/min) was >30-fold slower than the rate of SR-BI-dependent selective cholesteryl ester (CE) uptake ( approximately 17 ng HDL/mg/min), whereas the rate of release of HDL from the cell surface ( approximately 19 ng HDL/mg/min) was similar to the rate of selective CE uptake. Notably, the rate of SR-BI-dependent HDL resecretion in COS-7 and HepG2 cells was similar. BLT1, a compound that inhibits selective CE uptake, does not alter the amount of SR-BI-mediated HDL retroendocytosis in COS-7 cells. From these data, we conclude that HDL retroendocytosis in COS-7 and HepG2 cells is similar and that the vast majority of SR-BI-dependent selective uptake occurs at the cell surface in both cell types.
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Endocitosis/fisiología , Fibroblastos/metabolismo , Hepatocitos/metabolismo , Lipoproteínas HDL/metabolismo , Receptores Depuradores de Clase B/metabolismo , Animales , Biotinilación , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Ésteres del Colesterol/metabolismo , Humanos , Metabolismo de los Lípidos , Ratones , Microscopía Confocal , Receptores Depuradores de Clase B/genética , Receptores Depuradores de Clase B/fisiologíaRESUMEN
The high density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediates selective cholesteryl ester uptake from lipoproteins into liver and steroidogenic tissues but also cholesterol efflux from macrophages to HDL. Recently, we demonstrated the uptake of HDL particles in SR-BI overexpressing Chinese hamster ovarian cells (ldlA7-SRBI) using ultrasensitive microscopy. In this study we show that this uptake of entire HDL particles is followed by resecretion. After uptake, HDL is localized in endocytic vesicles and organelles en route to the perinuclear area; many HDL-positive compartments were classified as multivesiculated and multilamellated organelles by electron microscopy. By using 125I-labeled HDL, we found that approximately 0.8% of the HDL added to the media is taken up by the ldlA7-SRBI cells within 1 h, and almost all HDL is finally resecreted. 125I-Labeled low density lipoprotein showed a very similar association, uptake, and resecretion pattern in ldlA7-SRBI cells that do not express any low density lipoprotein receptor. Moreover, we demonstrate that the process of HDL cell association, uptake, and resecretion occurs in three physiologically relevant cell systems, the liver cell line HepG2, the adrenal cell line Y1BS1, and phorbol myristate acetate-differentiated THP-1 cells as a model for macrophages. Finally, we present evidence that HDL retroendocytosis represents one of the pathways for cholesterol efflux.
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Antígenos CD36/fisiología , Lipoproteínas HDL/metabolismo , Animales , Antígenos CD36/metabolismo , Células CHO , Línea Celular , Cloroquina/farmacología , Colesterol/química , Colesterol/metabolismo , Ésteres del Colesterol/metabolismo , Coloides/química , Cricetinae , Relación Dosis-Respuesta a Droga , Endocitosis , Endosomas/metabolismo , Exocitosis , Citometría de Flujo , Humanos , Lipoproteínas/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Microscopía Electrónica , Microscopía Fluorescente , Unión Proteica , Factores de TiempoRESUMEN
The high density lipoprotein (HDL) receptor Scavenger Receptor BII (SR-BII) is encoded by an alternatively spliced mRNA from the SR-BI gene and is expressed in various tissues. SR-BII protein differs from SR-BI only in the carboxyl-terminal cytoplasmic tail, which, as we showed previously, must contain a signal that confers predominant intracellular expression and rapid endocytosis of HDL. We have shown that SR-BII mediates HDL endocytosis through aclathrin-dependent, caveolae-independent pathway. Two candidate amino acid motifs were identified in the tail that could mediate association with clathrin-containing endocytic vesicles: a putative dileucine motif at position 492-493 and an overlapping tyrosine-based YXXZ motif starting at position 489. Although substitution of tyrosine at position 489 with alanine or histidine did not affect endocytosis, substitution L492A resulted in increased surface binding of HDL and reduced HDL particle endocytosis. Substitution L493A had a less dramatic effect. No other regions in the carboxyl-terminal tail appeared to contain motifs required for HDL endocytosis. Substitutions of leucine at position 492 with the hydrophobic amino acids valine or phenylalanine also reduced HDL endocytosis, stressing the importance of leucine at this position. Introducing the SR-BII YTPLL motif into the carboxyl-terminal cytoplasmic tail of SR-BI converted SR-BI into an endocytic receptor resembling SR-BII. These results demonstrated that SR-BII differs from SR-BI in subcellular localization and trafficking and suggest that the two isoforms differ in the manner in which they target ligands intracellularly.
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Clatrina/fisiología , Endocitosis , Lipoproteínas HDL/metabolismo , Proteínas de Membrana de los Lisosomas/fisiología , Receptores Depuradores/fisiología , Sialoglicoproteínas/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Humanos , Leucina , Proteínas de Membrana de los Lisosomas/química , Datos de Secuencia Molecular , Sialoglicoproteínas/químicaRESUMEN
Scavenger receptor class B, type I (SR-BI) mediates selective uptake of high density lipoprotein (HDL) lipids. It is unclear whether this process occurs at the cell membrane or via endocytosis. Our group previously identified an alternative mRNA splicing variant of SR-BI, named SR-BII, with an entirely different, yet highly conserved cytoplasmic C terminus. In this study we aimed to compare HDL uptake by both isoforms. Whereas SR-BI was mainly ( approximately 70%) localized on the surface of transfected Chinese hamster ovary cells, as determined by biotinylation, HDL binding at 4 degrees C, and studies of enhanced green fluorescent protein-tagged SR-BI/II fusion proteins, the majority of SR-BII ( approximately 80-90%) was expressed intracellularly. The cellular distribution of SR-BI was not affected by deletion of the C terminus, which suggests that the distinct C terminus of SR-BII is responsible for its intracellular expression. Pulse-chase experiments showed that SR-BII rapidly internalized HDL protein, whereas in the case of SR-BI most HDL protein remained surface bound. Like its ligand, SR-BII was more rapidly endocytosed compared with SR-BI. Despite more rapid HDL uptake by SR-BII than SR-BI, selective cholesteryl ether uptake was significantly lower. Relative to their levels of expression at the cell surface, however, both isoforms mediated selective uptake with similar efficiency. HDL protein that was internalized by SR-BII largely co-localized with transferrin in the endosomal recycling compartment. Within the endosomal recycling compartment of SR-BII cells, there was extensive co-localization of internalized HDL lipid and protein. These results do not support a model that selective lipid uptake by SR-BI requires receptor/ligand recycling within the cell. We conclude that SR-BII may influence cellular cholesterol trafficking and homeostasis in a manner that is distinct from SR-BI.
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HDL-Colesterol/farmacocinética , Proteínas de la Membrana , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Sialoglicoproteínas , Empalme Alternativo , Animales , Células CHO , Células COS , Cricetinae , Endocitosis/fisiología , Endosomas/metabolismo , Homeostasis/fisiología , Humanos , Ligandos , Proteínas de Membrana de los Lisosomas , Receptores Depuradores , Transfección , Transferrina/farmacocinéticaRESUMEN
Scavenger receptor class B type I (SR-BI) mediates selective uptake of cholesteryl esters from HDL as well as efflux of cellular free cholesterol to HDL. It is unclear whether the receptor is involved in intestinal cholesterol absorption. We addressed this issue by studying [3H]cholesterol flux in differentiated CaCo-2 cells incubated at their apical side with mixed taurocholate/phosphatidylcholine/cholesterol micelles. Biotinylation and HDL binding experiments showed predominant apical expression of endogenous and overexpressed SR-BI. Mixed micellar cholesterol saturation affected the magnitude and direction of cholesterol flux with significant net uptake only from supersaturated micelles and net efflux from unsaturated micelles. Incubation with micelles that depleted cellular cholesterol resulted in a decrease of SR-BI protein, whereas incubation with cholesterol-loading micelles resulted in a significant increase of SR-BI protein. Apical cholesterol uptake by CaCo-2 cells was increased in the presence of a SR-BI-blocking antibody and by partial inhibition of SR-BI expression with small inhibitory RNA. Adenovirus-mediated overexpression of apical SR-BI did not affect cholesterol uptake but stimulated apical cholesterol efflux, even to supersaturated mixed micelles. Partial inhibition of SR-BI with small inhibitory RNA reduced apical cholesterol efflux. Our data argue against a direct role for SR-BI in micellar cholesterol uptake. However, SR-BI might be involved in cholesterol absorption by facilitating cholesterol efflux to micelles.
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HDL-Colesterol/metabolismo , Colesterol/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Adenoviridae/genética , Anticuerpos/farmacología , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Antígenos CD36 , Células CACO-2 , Diferenciación Celular/fisiología , Polaridad Celular/fisiología , Colesterol/química , Enterocitos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Micelas , ARN Interferente Pequeño/genética , Receptores Inmunológicos/inmunología , Receptores Depuradores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptores Depuradores de Clase BRESUMEN
BACKGROUND & AIMS: In humans, cholesterol absorbed from the intestine contributes appreciably to serum cholesterol levels. We hypothesized that cholesterol thermodynamic activity (A(t)) would predict bioavailability of cholesterol monomers in intestinal content, and that natural dietary phospholipids exhibiting high affinity for cholesterol would reduce its absorption. METHODS: Cholesterol A(t) was determined by measuring partitioning of monomeric cholesterol from aqueous solutions of taurocholate, cholesterol, and either milk sphingomyelin (MSM), dipalmitoyl phosphatidylcholine (DPPC), or egg yolk phosphatidylcholine (EYPC) into wafers of polymerized silicone. Cholesterol absorption from the same mixtures was tested with monolayers of Caco-2 cells. For in vivo absorption studies (employing male C57L/J mice), we used the fecal dual isotope method during dietary enrichment with MSM, DPPC, or EYPC at varying dose levels. RESULTS: Cholesterol A(t) values were reduced significantly in MSM- and DPPC-containing systems compared with EYPC and correlated positively with reduced uptake and esterification of cholesterol by Caco-2 cells. Mice fed chow absorbed 31.4% +/- 6.9% (mean +/- SEM) cholesterol, whereas enrichment with MSM or DPPC led to dose-dependent decreases in cholesterol absorption; even at 0.1% MSM, cholesterol absorption was reduced by 20.4% +/- 15.4% (P < 0.05, n = 6). CONCLUSIONS: Different phospholipids have distinct effects on micellar cholesterol A(t), which predicts cholesterol uptake by enterocytes in vitro as well as in vivo. Natural phospholipids with high affinity for cholesterol, as evidenced particularly by sphingomyelin, decrease A(t) and curtail intestinal cholesterol absorption.