Iron-ascorbate alters the efficiency of Caco-2 cells to assemble and secrete lipoproteins

Courtois, Frédéric; Suc, Isabelle; Garofalo, Carole; Ledoux, M.; Seidman, Ernest G.; Lévy, Émile

doi:10.1152/ajpgi.2000.279.1.g12

Cited by 43 publications

(39 citation statements)

References 42 publications

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“…Malondialdehyde (MDA)-The amount of MDA was determined by HPLC as described previously (28). Briefly, proteins were first precipitated with a 10% sodium tungstate (Na 2 WO 4 ) (Aldrich) solution, and protein-free supernatant was then reacted with an isovolume of 0.5% thiobarbituric acid (Sigma) solution at 90°C for 60 min.…”

Section: Estimation Of Lipid Peroxidationmentioning

confidence: 99%

Modification in Oxidative Stress, Inflammation, and Lipoprotein Assembly in Response to Hepatocyte Nuclear Factor 4α Knockdown in Intestinal Epithelial Cells

Marcil

Seidman

Sinnett

et al. 2010

Journal of Biological Chemistry

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Section: Estimation Of Lipid Peroxidationmentioning

confidence: 99%

Modification in Oxidative Stress, Inflammation, and Lipoprotein Assembly in Response to Hepatocyte Nuclear Factor 4α Knockdown in Intestinal Epithelial Cells

Marcil

Seidman

Sinnett

et al. 2010

Journal of Biological Chemistry

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“…For individual experiments, cells were plated at a density of 1 3 10 6 cells/well on 24.5 mm polycarbonate Transwell filter inserts with 0.4 mm pores (Costar, Cambridge, MA) in DMEM (as described above) supplemented with 5% FBS. The inserts were placed onto six-well culture plates and cultured for 20 days, at which time Caco-2 cells are highly differentiated and appropriate for lipid metabolism (23,24).…”

Section: Cell Culturementioning

confidence: 99%

“…Enzymatic activity was assayed as described previously (24,26 14 C]mevalonate formed was converted into lactone by the addition of 10 N HCl, isolated by TLC, and counted using an internal standard to correct for incomplete recovery.…”

Section: Hmg-coa Reductase Activity Assaymentioning

confidence: 99%

Localization and role of NPC1L1 in cholesterol absorption in human intestine

Sané

Sinnett

Delvin

et al. 2006

Journal of Lipid Research

144

122

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Recent studies have documented the presence of Niemann-Pick C1-Like 1 (NPC1L1) in the small intestine and its capacity to transport cholesterol in mice and rats. The current investigation was undertaken to explore the localization and function of NPC1L1 in human enterocytes. Cell fractionation experiments revealed an NPC1L1 association with apical membrane of the enterocyte in human jejunum. Signal was also detected in lysosomes, endosomes, and mitochondria. Confirmation of cellular NPC1L1 distribution was obtained by immunocytochemistry. Knockdown of NPC1L1 caused a decline in the ability of Caco-2 cells to capture micellar [14 C]free cholesterol. Furthermore, this NPC1L1 suppression resulted in increased and decreased mRNA levels and activity of HMG-CoA reductase, the rate-limiting step in cholesterol synthesis, and of ACAT, the key enzyme in cholesterol esterification, respectively. An increase was also noted in the transcriptional factor sterol-regulatory element binding protein that modulates cholesterol homeostasis. Efforts were devoted to define the impact of NPC1L1 knockdown on other mediators of cholesterol uptake. RT-PCR evidence is presented to show the significant decrease in the levels of scavenger receptor class B type I (SR-BI) with no changes in ABCA1, ABCG5, and cluster determinant 36 in NPC1L1-deficient Caco-2 cells. Together, our data suggest that NPC1L1 contributes to intestinal cholesterol homeostasis and possibly cooperates with SR-BI to mediate cholesterol absorption in humans. Coronary heart disease is the most important clinical manifestation of atherosclerosis and remains the main cause of death in developed societies. Hypercholesterolemia is beyond doubt the most prominent risk factor for the development of atherosclerosis, which is caused by derangements in cholesterol homeostasis (i.e., intestinal uptake, endogenous synthesis and metabolism, transport in lipoprotein particles, and biliary excretion). At present, there are increasing efforts to understand the physiology of intestinal fat transport in view of the positive relationship between cholesterol absorption, plasma cholesterol levels, and coronary heart disease (1-4). Additionally, augmented dietary fat and cholesterol intake have been tightly linked to the increased incidence of other diseases, such as cancer, diabetes, and obesity (5-7).In stark contrast to previous tenets suggesting that cholesterol uptake occurs as a passive diffusion down a concentration gradient, more and more investigators now support protein-mediated cholesterol absorption. Curiously, protein transporters intervening in intestinal cholesterol movement have not yet been ascertained. A few recent studies have proposed scavenger receptor class B type I (SR-BI) as a candidate protein involved in dietary cholesterol transport (8-11). On the other hand, the involvement of SR-BI in cholesterol uptake has been questioned because intestinal cholesterol absorption was shown to be unaffected by deletion of the SR-BI gene in mice (10, 12). Other laboratori...

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“…For individual experiments, cells were plated at a density of 1×10 6 cells/well on 24.5 mm polycarbonate transwell filter inserts with 0.4 µm pores (Costar, Cambridge, MA) in MEM (as described above) supplemented with 5% FBS. The inserts were placed into six-well culture plates and cultured for 20 days, a period at which the Caco-2 cells are highly differentiated and appropriate for lipid metabolism (Marcil et al, 2002;Courtois et al, 2000).…”

Section: Cell Culturementioning

confidence: 99%

Ontogeny, immunolocalisation, distribution and function of SR-BI in the human intestine

Lévy

Ménard

Suc

et al. 2004

Journal of Cell Science

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Studies employing human fetal intestine have yielded remarkable information on the role of polarized enterocytes in fat absorption. In this report, we investigated the intestinal expression, spatiotemporal distributions, ontogeny and function of the scavenger receptor, Class B, Type I (SR-BI) that plays a crucial role in cholesterol homeostasis. SR-BI was detected as early as week 14 of gestation in all gut segments and was almost entirely confined to the absorptive epithelial cells. By using immunofluorescence staining, the distribution of SR-BI rarely appeared as a gradient, increasing from the developing crypt to the tip of the villus. Western blot showed high levels of immunodetectable SR-BI in the duodenum, which progressively decreased toward the distal colon. The high-resolution immunogold technique revealed labelling mainly over microvilli of the enterocyte. SR-BI was not associated with caveolin-1 and was not detectable in caveolae. In order to define the role of SR-BI in intestinal cholesterol absorption, Caco-2 cells were transfected with a constitutive expression vector (pZeoSV) containing human SR-BI cDNA inserted in an antisense orientation. As noted by immunoblotting and Protein A-gold techniques, stable transformants contained 40, 60 and 80% the SR-BI level of control Caco-2 cells and exhibited a proportional drop in free cholesterol uptake without altering the capture of phospholipids or cholesteryl ester. Confirmation of these data was obtained in intestinal organ culture where SR-BI antibodies lowered cholesterol uptake. These observations suggest that the human intestine possesses a developmental and regional SR-BI pattern of distribution, and extends our knowledge in SR-BI-mediated cholesterol transport.

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Iron-ascorbate alters the efficiency of Caco-2 cells to assemble and secrete lipoproteins

Cited by 43 publications

References 42 publications

Modification in Oxidative Stress, Inflammation, and Lipoprotein Assembly in Response to Hepatocyte Nuclear Factor 4α Knockdown in Intestinal Epithelial Cells

Modification in Oxidative Stress, Inflammation, and Lipoprotein Assembly in Response to Hepatocyte Nuclear Factor 4α Knockdown in Intestinal Epithelial Cells

Localization and role of NPC1L1 in cholesterol absorption in human intestine

Ontogeny, immunolocalisation, distribution and function of SR-BI in the human intestine

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