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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Tolerance to morphine analgesia is believed to result from a neuronal adaptation produced by continuous drug administration, although the precise mechanisms involved have yet to be established. Recently, we reported selective alterations in rat spinal calcitonin gene-related peptide (CGRP) markers in morphine-tolerant animals. In fact, increases in CGRP-like immunostaining and decrements in specific [125]hCGRP binding in the superficial laminae of the dorsal horn were correlated with the development of tolerance to the spinal antinociceptive action of morphine. Other spinally located peptides such as substance P, galanin, and neuropeptide Y were unaffected. Thus, the major goal of the present study was to investigate whether the development of tolerance to spinally infused morphine could be modulated by the blockade of dorsal horn CGRP receptors using the potent CGRP antagonist hCGRP(8-37). Indeed, cotreatments with hCGRP(8-37) prevented, in a dose-dependent manner, the development of tolerance to morphine-induced analgesia in both the rat tail-flick/tail-immersion and paw-pressure tests. Moreover, alterations in spinal CGRP markers seen in morphine-tolerant animals were not observed after a coadministration of morphine and hCGRP(8-37). These results demonstrate the existence of specific interaction between CGRP and the development of tolerance to the spinal antinociceptive effects of morphine. They also suggest that CGRP receptor antagonists could become useful adjuncts in the treatment of pain and tolerance to the antinociceptive effects of morphine.

1 Opioid receptors in the brain activate descending pain pathways to inhibit the nociceptive response to acute noxious stimuli. The aim of the present study was to clarify the role of supraspinal opioid receptors in modulating the nociceptive response to persistent in¯ammation in rats. 2 Subcutaneous administration of 50 ml of complete Freund's Adjuvant (CFA) into the plantar surface of the hindpaw induced a signi®cant decrease in paw withdrawal latency to thermal stimuli (P50.01) at 24 h post-injection. 3 Intracerebroventricular (i.c.v.) administration of the m opioid receptor agonists, DAMGO and morphine, and the d opioid receptor agonists, deltorphin II and SNC80, signi®cantly reversed the hyperalgesic response associated with peripheral in¯ammation in a dose-dependent manner (P50.0001). 4 The m and d agonists also signi®cantly attenuated the antinociceptive response to acute thermal stimulation in rats (P50.001). However, deltorphin II and SNC80 were less potent, and in the case of SNC80 less e cacious, in modulating the response to acute thermal nociception in comparison to hyperalgesia associated with persistent in¯ammation. 5 These results indicate that m and d opioid receptors in the brain modulate descending pain pathways to attenuate the nociceptive response to acute thermal stimuli in both normal and in¯amed tissues. The heightened response to d agonists in the hyperalgesia model suggests that d opioid receptors in the brain are promising targets for the treatment of pain arising from chronic in¯ammation.

Based on titration microcalorimetry and Caco-2 cell line transfection studies, it has been suggested that the A54T of the FABP2 gene plays a significant role in the assimilation of dietary fatty acids. However, reports were divergent with regard to the in vivo interaction between this polymorphism and postprandial lipemia. We therefore determined the influence of this intestinal fatty acid-binding protein polymorphism on intestinal fat transport using the human jejunal organ culture model, thus avoiding the interference of various circulating factors capable of metabolizing in vivo postprandial lipids. Analysis of DNA samples from 32 fetal intestines revealed 22 homozygotes for the wild-type Ala-54/ Ala-54 genotype (0.83) and 10 heterozygotes for the polymorphic Thr-54/Ala-54 genotype (0.17). The Thr-encoding allele was associated with increased secretion of newly esterified triglycerides, augmented de novo apolipoprotein B synthesis, and elevated chylomicron output. On the other hand, no alterations were found in very low density lipoprotein and high density lipoprotein production, apolipoprotein A-I biogenesis, or microsomal triglyceride transfer protein mass and activity. Similarly, the alanine to threonine substitution at residue 54 did not result in changes in brush border hydrolytic activities (sucrase, glucoamylase, lactase, and alkaline phosphatase) or in glucose uptake or oxidation. Our data clearly document that the A54T polymorphism of FABP2 specifically influences small intestinal lipid absorption without modifying glucose uptake or metabolism. It is proposed that, in the absence of confounding factors such as environmental and genetic variables, the FABP2 polymorphism has an important effect on postprandial lipids in vivo, potentially influencing plasma levels of lipids and atherogenesis.

SUMMARY We investigated the spatiotemporal distributions of the different peroxisome proliferator-activated receptor (PPAR) isotypes (α, β, and α) during development (Week 7 to Week 22 of gestation) of the human fetal digestive tract by immunohistochemistry using specific polyclonal antibodies. The PPAR subtypes, including PPARα, are expressed as early as 7 weeks of development in cell types of endodermal and mesodermal origin. The presence of PPARα was also found by Western blotting and nuclease-S1 protection assay, confirming that this subtype is not adipocyte-specific. PPARα, PPARβ, and PPARα exhibit different patterns of expression during morphogenesis of the digestive tract. Whatever the stage and the gut region (except the stomach) examined, PPARα is expressed at a high level, suggesting some fundamental role for this receptor in development and/or physiology of the human digestive tract.

cDNA microarray technology enables detailed analysis of gene expression throughout complex processes such as differentiation. The aim of this study was to analyze the gene expression profile of normal human intestinal epithelial cells using cell models that recapitulate the crypt-villus axis of intestinal differentiation in comparison with the widely used Caco-2 cell model. cDNA microarrays (19,200 human genes) and a clustering algorithm were used to identify patterns of gene expression in the crypt-like proliferative HIEC and tsFHI cells, and villus epithelial cells as well as Caco-2/15 cells at two distinct stages of differentiation. Unsupervised hierarchical clustering analysis of global gene expression among the cell lines identified two branches: one for the HIEC cells versus a second comprised of two sub-groups: (a) the proliferative Caco-2 cells and (b) the differentiated Caco-2 cells and closely related villus epithelial cells. At the gene level, supervised hierarchical clustering with 272 differentially expressed genes revealed distinct expression patterns specific to each cell phenotype. We identified several upregulated genes that could lead to the identification of new regulatory pathways involved in cell differentiation and carcinogenesis. The combined use of microarray analysis and human intestinal cell models thus provides a powerful tool for establishing detailed gene expression profiles of proliferative to terminally differentiated intestinal cells. Furthermore, the molecular differences between the normal human intestinal cell models and Caco-2 cells clearly point out the strengths and limitations of this widely used experimental model for studying intestinal cell proliferation and differentiation.

Although intestinal (I) and liver (L) fatty acid binding proteins (FABP) have been widely studied, the physiological significance of the presence of the two FABP forms (I- and L-FABP) in absorptive cells remains unknown as do the differences related to their distribution along the crypt-villus axis, regional expression, ontogeny and regulation in the human intestine. Our morphological experiments supported the expression of I- and L-FABP as early as 13 weeks of gestation. Whereas cytoplasmic immunofluorescence staining of L-FABP was barely detectable in the lower half of the villus and in the crypt epithelial cells, I-FABP was visualized in epithelial cells of the crypt-villus axis in all intestinal segments until the adult period in which the staining was maximized in the upper part of the villus. Immunoelectron microscopy revealed more intense labeling of L-FABP compared with I-FABP, accompanied with a heterogeneous distribution in the cytoplasm, microvilli and basolateral membranes. By western blot analysis, I- and L-FABP at 15 weeks of gestation appeared predominant in jejunum compared with duodenum, ileum, proximal and distal colon. Exploration of the maturation aspect documented a rise in L-FABP in adult tissues. Permanent transfections of Caco-2 cells with I-FABP cDNA resulted in decreased lipid export, apolipoprotein (apo) biogenesis and chylomicron secretion. Additionally, supplementation of Caco-2 with insulin, hydrocortisone and epidermal growth factor differentially modulated the expression of I- and L-FABP, apo B-48 and microsomal triglyceride transfer protein (MTP), emphasizing that these key proteins do not exhibit a parallel modulation. Overall, our findings indicate that the two FABPs display differences in localization, regulation and developmental pattern.