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Localization and role of NPC1L1 in cholesterol absorption in human intestine

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 [14C]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.

Mechanisms of lipid malabsorption in Cystic Fibrosis: the impact of essential fatty acids deficiency

Transport mechanisms, whereby alimentary lipids are digested and packaged into small emulsion particles that enter intestinal cells to be translocated to the plasma in the form of chylomicrons, are impaired in cystic fibrosis. The purpose of this paper is to focus on defects that are related to intraluminal and intracellular events in this life-limiting genetic disorder. Specific evidence is presented to highlight the relationship between fat malabsorption and essential fatty acid deficiency commonly found in patients with cystic fibrosis that are often related to the genotype. Given the interdependency of pulmonary disease, pancreatic insufficiency and nutritional status, greater attention should be paid to the optimal correction of fat malabsorption and essential fatty acid deficiency in order to improve the quality of life and extend the life span of patients with cystic fibrosis.

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

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.

Prevention of oxidative stress, inflammation and mitochondrial dysfunction in the intestine by different cranberry phenolic fractions

Cranberry fruit has been reported to have high antioxidant effectiveness that is potentially linked to its richness in diversified polyphenolic content. The aim of the present study was to determine the role of cranberry polyphenolic fractions in oxidative stress (OxS), inflammation and mitochondrial functions using intestinal Caco-2/15 cells. The combination of HPLC and UltraPerformance LC®-tandem quadrupole (UPLC-TQD) techniques allowed us to characterize the profile of low, medium and high molecular mass polyphenolic compounds in cranberry extracts. The medium molecular mass fraction was enriched with flavonoids and procyanidin dimers whereas procyanidin oligomers (DP > 4) were the dominant class of polyphenols in the high molecular mass fraction. Pre-incubation of Caco-2/15 cells with these cranberry extracts prevented iron/ascorbate-mediated lipid peroxidation and counteracted lipopolysaccharide-mediated inflammation as evidenced by the decrease in pro-inflammatory cytokines (TNF-α and interleukin-6), cyclo-oxygenase-2 and prostaglandin E2. Cranberry polyphenols (CP) fractions limited both nuclear factor κB activation and Nrf2 down-regulation. Consistently, cranberry procyanidins alleviated OxS-dependent mitochondrial dysfunctions as shown by the rise in ATP production and the up-regulation of Bcl-2, as well as the decline of protein expression of cytochrome c and apoptotic-inducing factor. These mitochondrial effects were associated with a significant stimulation of peroxisome-proliferator-activated receptor γ co-activator-1-α, a central inducing factor of mitochondrial biogenesis and transcriptional co-activator of numerous downstream mediators. Finally, cranberry procyanidins forestalled the effect of iron/ascorbate on the protein expression of mitochondrial transcription factors (mtTFA, mtTFB1, mtTFB2). Our findings provide evidence for the capacity of CP to reduce intestinal OxS and inflammation while improving mitochondrial dysfunction.

Interactions Between the Dietary Polyunsaturated Fatty Acid Ratio and Genetic Factors Determine Susceptibility to Pediatric Crohn's Disease

Increased dietary ratios of ω6/ω3 polyunsaturated fatty acids have been implicated in the pathogenesis of Crohns disease (CD), but epidemiologic data are limited. We investigated whether variants of genes that control polyunsaturated fatty acid metabolism (CYP4F3, FADS1, and FADS2), along with the dietary ratio of ω6/ω3, confers susceptibility to CD. Based on data from 182 children newly diagnosed with CD and 250 controls, we found that children who consumed a higher dietary ratio of ω6/ω3 were susceptible for CD if they were also carriers of specific variants of CYP4F3 and FADS2 genes. Our findings implicate diet-gene interactions in the pathogenesis of CD.

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

Hepatocyte nuclear factor 4α (HNF4α) is a nuclear transcription factor mainly expressed in the liver, intestine, kidney, and pancreas. Many of its hepatic and pancreatic functions have been described, but limited information is available on its role in the gastrointestinal tract. The objectives of this study were to evaluate the anti-inflammatory and antioxidant functions of HNF4α as well as its implication in intestinal lipid transport and metabolism. To this end, the HNF4A gene was knocked down by transfecting Caco-2 cells with a pGFP-V-RS lentiviral vector containing an shRNA against HNF4α. Inactivation of HNF4α in Caco-2 cells resulted in the following: (a) an increase in oxidative stress as demonstrated by the levels of malondialdehyde and conjugated dienes; (b) a reduction in secondary endogenous antioxidants (catalase, glutathione peroxidase, and heme oxygenase-1); (c) a lower protein expression of nuclear factor erythroid 2-related factor that controls the antioxidant response elements-regulated antioxidant enzymes; (d) an accentuation of cellular inflammatory activation as shown by levels of nuclear factor-κB, interleukin-6, interleukin-8, and leukotriene B4; (e) a decrease in the output of high density lipoproteins and of their anti-inflammatory and anti-oxidative components apolipoproteins (apo) A-I and A-IV; (f) a diminution in cellular lipid transport revealed by a lower cellular secretion of chylomicrons and their apoB-48 moiety; and (g) alterations in the transcription factors sterol regulatory element-binding protein 2, peroxisome proliferator-activated receptor α, and liver X receptor α and β. In conclusion, HNF4α appears to play a key role in intestinal lipid metabolism as well as intestinal anti-oxidative and anti-inflammatory defense mechanisms.

Expression of Sar1b Enhances Chylomicron Assembly and Key Components of the Coat Protein Complex II System Driving Vesicle Budding

Objective—SAR1b plays a significant role in the assembly, organization, and function of the coat protein complex II, a critical complex for the transport of proteins from the endoplasmic reticulum to the Golgi. Recently, mutations in SARA2 have been associated with lipid absorption disorders. However, functional studies on Sar1b-mediated lipid synthesis pathways and lipoprotein packaging have not been performed. Methods and Results—Sar1b was overexpressed in Caco-2/15 cells and resulted in significantly augmented triacylglycerol, cholesteryl ester, and phospholipid esterification and secretion and markedly enhanced chylomicron production. It also stimulated monoacylglycerol acyltransferase/diacylglycerol acyltransferase activity and enhanced apolipoprotein B-48 protein synthesis, as well as elevated microsomal triglyceride transfer protein activity. Along with the enhanced chylomicrons, microsomes were characterized by abundant Sec12, the guanine exchange factor that promotes the localization of Sar1b in the endoplasmic reticulum. Furthermore, coimmunoprecipitation experiments revealed high levels of the complex components Sec23/Sec24 and p125, the Sec23-interacting protein. Finally, a pronounced interaction of Sec23/Sec24 with sterol regulatory element binding protein (SREBP) cleavage-activating protein and SREBP-1c was noted, thereby permitting the transfer of the transcription factor SREBP-1c to the nucleus for the activation of genes involved in lipid metabolism. Conclusion—Our data suggest that Sar1b expression may promote intestinal lipid transport with the involvement of the coat protein complex II network and the processing of SREBP-1c.

Iron-ascorbate-mediated lipid peroxidation causes epigenetic changes in the antioxidant defense in intestinal epithelial cells: impact on inflammation.

Introduction The gastrointestinal tract is frequently exposed to noxious stimuli that may cause oxidative stress, inflammation and injury. Intraluminal pro-oxidants from ingested nutrients especially iron salts and ascorbic acid frequently consumed together, can lead to catalytic formation of oxygen-derived free radicals that ultimately overwhelm the cellular antioxidant defense and lead to cell damage. Hypothesis Since the mechanisms remain sketchy, efforts have been exerted to evaluate the role of epigenetics in modulating components of endogenous enzymatic antioxidants in the intestine. To this end, Caco-2/15 cells were exposed to the iron-ascorbate oxygen radical-generating system. Results Fe/Asc induced a significant increase in lipid peroxidation as reflected by the elevated formation of malondialdehyde along with the alteration of antioxidant defense as evidenced by raised superoxide dismutase 2 (SOD2) and diminished glutathione peroxidase (GPx) activities and genes. Consequently, there was an up-regulation of inflammatory processes illustrated by the activation of NF-κB transcription factor, the higher production of interleukin-6 and cycloxygenase-2 as well as the decrease of IκB. Assessment of promoter’s methylation revealed decreased levels for SOD2 and increased degree for GPx2. On the other hand, pre-incubation of Caco-2/15 cells with 5-Aza-2′-deoxycytidine, a demethylating agent, or Trolox antioxidant normalized the activities of SOD2 and GPx, reduced lipid peroxidation and prevented inflammation. Conclusion Redox and inflammatory modifications in response to Fe/Asc -mediated lipid peroxidation may implicate epigenetic methylation.

Genome-wide association study signal at the 12q12 locus for Crohn's disease may represent associations with the MUC19 gene.

Background:Genome-wide association studies (GWAS) in Crohn’s disease (CD) have identified associations with single-nucleotide polymorphism (SNP) rs11175593 at chromosome 12q12. The MUC19 and LRRK2 genes reside close to the GWAS signal, but it is as yet unclear which of the 2 genes represent the CD susceptibility genes. Methods:We studied associations between nonsynonymous coding variants in the MUC19 (5) and LRRK2 (3) genes in a case–control sample comprising CD cases aged <18 years at diagnosis. The GWAS lead SNP rs11175593 was also investigated. Allelic, genotype, and haplotype associations were examined assuming different models of inheritance. Results:A total of 530 cases and 600 controls were studied. The mean (±SD) age at diagnosis was 12.4 (±3.3). Most cases were male (57.4%). Most patients had ileocolonic disease location (48.8%) and inflammatory behavior at diagnosis (87.0%). Three MUC19 SNPs were nominally significantly associated with CD (rs11564245, Asp→His: P = 0.02; rs4768261, Ser→Phe: P = 0.0008; and rs2933353, Glu→Ala: P = 0.01). Associations with rs4768261 were maintained after corrections for multiple comparisons (permuted, P = 0.007). None of the LRRK2 SNPs were associated with CD. Haplotype analysis supported the single SNP associations noted with the MUC19 gene. Conclusions:GWAS signal at chromosome 12q12 for CD may represent associations with the MUC19 gene.

Sar1b transgenic male mice are more susceptible to high-fat diet-induced obesity, insulin insensitivity and intestinal chylomicron overproduction ☆

In the intracellular secretory network, nascent proteins are shuttled from the endoplasmic reticulum to the Golgi by transport vesicles requiring Sar1b, a small GTPase. Mutations in this key enzyme impair intestinal lipid transport and cause chylomicron retention disease. The main aim of this study was to assess whether Sar1b overexpression under a hypercaloric diet accelerated lipid production and chylomicron (CM) secretion, thereby inducing cardiometabolic abnormalities. To this end, we generated transgenic mice overexpressing human Sar1b (Sar1b(+/+)) using pBROAD3-mcs that features the ubiquitous mouse ROSA26 promoter. In response to a high-fat diet (HFD), Sar1b(+/+) mice displayed significantly increased body weight and adiposity compared with Sar1b(+/+) mice under the same regimen or with wild-type (WT) mice exposed to chow diet or HFD. Furthermore, Sar1b(+/+) mice were prone to liver steatosis as revealed by significantly elevated hepatic triglycerides (TG) and cholesterol in comparison with WT animals. They also exhibited augmented levels of plasma TG along with alterations in fatty acid composition. Concomitantly, they showed susceptibility to develop insulin insensitivity and they responded abnormally to oral glucose tolerance test. Finally, Sar1b(+/+) mice that have been treated with Triton WR-1330 (to inhibit TG catabolism) and orotic acid (to block secretion of very low-density lipoprotein by the liver) responded more efficiently to fat meal tests as reflected by the rise in plasma TG and CM concentrations, indicating exaggerated intestinal fat absorption. These results suggest that Sar1b(+/+) under HFD can elicit cardiometabolic traits as revealed by incremental weight gain, fat deposition, dyslipidemia, hepatic steatosis, insulin insensitivity and intestinal fat absorption.