Departments of 3 Pediatrics, 2 Biochemistry, and 1 Nutrition, Université de Montréal, H3C 3J7; and 4 Department of Anatomy and Cellular Biology, Université de Sherbrooke, Montreal, Quebec, Canada H3T 1C5
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ABSTRACT |
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To examine the multiple stages of lipoprotein packaging during development, we studied localization, ontogeny, and regulation of microsomal transfer protein (MTP), a crucial protein for lipid transport. With the use of immunofluorescence, MTP was identified in villus and crypt epithelial cells in different regions of human fetal intestine, including colon. Staining was detected as early as the 13th wk of gestation in all gut segments and was almost entirely confined to the columnar epithelial cells of the jejunum and colon. Unlike immunofluorescence, which provides qualitative but not quantitative information on MTP signal, enzymatic assays revealed a decreasing gradient from proximal small intestine to distal, as confirmed by immunoblot. Activity of MTP in small intestinal explants cultured for different incubation periods (0, 4, 8, and 24 h) peaked at 4 h but remained insensitive to different concentrations of oleic acid. Also, a trend toward increasing MTP activity was observed at 20-22 wk of gestation. Finally, in strong contrast to jejunal efficiency, colonic explants displayed impaired lipid production, apolipoprotein biogenesis, and lipoprotein assembly, in association with poor expression of MTP. These findings provide the first evidence that human fetal gut is able to express MTP and emphasize the distinct regional distribution, regulation by oleic acid, and ontogeny of MTP.
fat absorption; developmental biology; lipoprotein metabolism; intestinal transport
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INTRODUCTION |
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CHYLOMICRON IS THE MAIN VEHICLE for the transport of dietary lipids and fat-soluble vitamins (4, 8, 45). It is exclusively synthesized in the intestine by a highly complex process that requires the translocation of apolipoprotein (apo) B into the lumen of the endoplasmic reticulum (10, 15, 25). Compelling evidence has now demonstrated the pivotal role of microsomal transfer protein (MTP) in the biogenesis of apo B-containing lipoproteins (42, 49). MTP is a soluble microsomal heterodimer consisting of the multifunctional 58-kDa protein disulfide isomerase (PDI) and a unique large 97-kDa protein that confers lipid transfer activity (48). Identification of MTP as the defective factor in abetalipoproteinemia indicates its essential function in intracellular apo B lipoprotein assembly (43, 48, 49). Despite knowledge of the morphological, ultrastructural, and functional changes occurring during the development of the human gut, information on the biosynthetic events essential for the formation and secretion of triglyceride-rich lipoprotein particles remains fragmentary. Our early studies (23, 24) have successfully used the organ culture technique not only to clarify the mechanism of intracellular lipid transport impairment in human disorders but also to explore the ontogeny and site of lipoprotein synthesis in the small intestine (28, 44). The maintenance of human fetal intestinal tissues in serum-free organ culture has allowed us to demonstrate that, between 14 and 20 wk of gestation, fetuses have functional mechanisms to elaborate all of the lipid classes and to secrete them in the form of lipoprotein particles (28, 44). Our findings have stressed the gradual increase in triglycerides, and chylomicrons in particular, as a function of gestational age, suggesting an ontogeny of the intracellular events leading to lipoprotein formation and exocytosis (44). We also provided evidence for de novo apolipoprotein synthesis, a process that has been proven to be hormonally controlled (26-28, 32, 33). The combination of immunocytofluorescence with radioactivity pulse labeling revealed the regulation of apo A-I and apo B by epidermal growth factor, hydrocortisone, and insulin (26-28, 32, 33). It is therefore clear that the mucosal phase of lipid absorption is intact in the developing intestine. However, it is evident that other probes need to be used to examine the assembly and secretion of apo B-containing lipoproteins during gestational periods. Although the role of MTP has never been studied in the prenatal gut mucosa, we surmised that the developing small intestinal epithelium expresses MTP because it secretes apo B in particles, largely of chylomicron, very low density lipoprotein, and low density lipoprotein size (28, 44). The essence of this hypothesis is that MTP is essential for the first step of apo B core lipidation, transferring sufficient amounts of triglycerides and cholesteryl esters into the hydrophobic domains of apo B to release apo B from endoplasmic reticulum membranes (the requisite first step in the assembly of triglyceride-rich lipoproteins) (20). Furthermore, the localization, ontogeny, and regulation of MTP were not investigated during the gestational period. In addition, the distribution profile of PDI has not been established in the small and large intestine during development. Finally, in our ongoing effort to further our understanding of the limited ability of the fetal colon to assemble and secrete apo B-containing lipoproteins (22, 29), we examined MTP activity to determine its implication in the defective exocytosis. In this study, we focused on all of these issues to gain insight into the multiple stages of lipoprotein packaging during development.
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MATERIALS AND METHODS |
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Human Gut Specimens
Small intestine and large bowel tissues were obtained from fetuses ranging from 13 to 20 wk following legal or therapeutic abortion with informed consent of the patients. No tissues were collected from cases associated with known fetal abnormalities or fetal death. Studies were approved by the Institutional Review Committee for the Use of Human Material of the Centre Hospitalier Universitaire de Sherbrooke. The entire gut was immersed in Leibovitz L-15 medium containing gentamicin (Garamycin, 40 µg/ml) and nystatin (Mycostatin, 40 µg/ml) and brought to the culture room within 30 min. The proximal half of the small intestine excluding the first 3 cm was defined as the jejunum, whereas the second part was defined as the ileum. The colon was divided in two equivalent portions, the proximal and distal parts.Organ Culture
This technique was carried out as described previously (26-29, 32, 33, 44). The intestine and colon were cleansed of mesentery, split longitudinally, washed in culture medium, and cut into explants (3 × 7 mm). Five to seven explants were randomly transferred onto lens paper with the mucosal side facing up in each organ culture dish (Falcon Plastics, Los Angeles, CA). An amount of medium (0.8 ml) sufficient to dampen the lens paper was added. Explants were cultured in serum-free Leibovitz L-15 medium according to the technique described previously (26-29, 32, 33, 35, 36, 44).Indirect Immunofluorescence
The preparation and embedding of specimens for cryosectioning were performed as previously described (2) using optimum cutting temperature embedding compounds (Tissue Tek; Miles Laboratories, Alkhart, IN). Cryosections (3-4 µm thick), cut on a Jung Frigocut 2800 N cryostat (Leica Canada, Saint-Laurent, PQ), were fixed on glass slides with acetone-chloroform (1:1) for 5 min at 4°C and washed twice with PBS. Following a 30-min incubation with 10% Blotto-PBS to quench the remaining aldehyde residues, the glass slides were washed twice in PBS. The staining procedure using antibodies and fluorescein was performed at room temperature in humid chambers. Sections were incubated for 1 h with the polyclonal antibodies (generously provided by Drs. J. R. Wetterau and H. Jamil) diluted in 10% Blotto (in PBS-8% BSA, 1:1) at 1:150 for MTP large subunit and PDI. Sections were then washed in PBS and incubated with fluorescein-conjugated sheep anti-rabbit IgG (Boehringer Mannheim, Laval, PQ, Canada) used at dilutions of 1:30 and 1:50 in 10% Blotto-PBS. After extensive washing with PBS, the sections were then mounted in FluoroGuard antipode (Bio-Rad) and viewed with a Reichert Polyvar 2 microscope (Leica, Montreal, PQ, Canada), equipped for epifluorescence. Finally, the primary antibodies were omitted or replaced by nonimmune rabbit serum at 1:700 dilution. All of these control experiments confirmed the specificity of the results.Tissue Preparation for Determination of MTP Activity
The fetal intestinal specimens were homogenized in 1 ml of 10 mM phosphate buffer (pH 6.8) containing saponin (100 µg/ml) and protease inhibitors (leupeptin 10 µg/ml, Trasylol 10 µg/ml, and pepstatin A 1 µg/ml). The MTP/PDI heterodimer was separated by ultracentrifugation at 100,000 g, and the supernatant was loaded onto a 1-ml DEAE cellulose column, preequilibrated with the same buffer (10 mM phosphate buffer, pH 6.8). Impurities were eluted with 2 ml of 10 mM phosphate buffer (pH 6.8) containing 30 mM NaCl. MTP/PDI heterodimer was eluted with 3 ml of 10 mM phosphate buffer (pH 6.8) containing 220 mM NaCl and concentrated using a Centricon 30 cartridge (5,000 rpm for 30 min).MTP Activity Assay
The triglyceride transfer assay was adapted from Refs. 20 and 51. MTP transfer activity was determined by evaluating the transfer of radiolabeled triacylglycerol between two populations of unilamellar vesicles, as described (19, 20, 51). The donor and acceptor vesicles were prepared by adding the appropriate amount of lipids to 500 µl of chloroform, followed by drying under a stream of nitrogen, rehydration, and probe sonication in 1.25 ml buffer (15 mM Tris · HCl, pH 7.4, 35 mM NaCl, 0.05% BSA, 3 mM sodium azide, and 1 mM EDTA). Donor vesicles contained (per assay) 4 nmol of egg yolk phosphatidylcholine, 0.33 nmol of cardiolipin, and 0.024 nmol of [3H]trioleylglycerol (Amersham). Acceptor vesicles contained 24 nmol of egg yolk phosphatidylcholine, 0.048 nmol of trioleylglycerol, and ~4,000 cpm [14C]dipalmitoyl phosphatidylcholine (Amersham). Both categories of vesicles comprised 0.01% butylated hydroxytoluene. Various amounts of semipurified MTP were incubated with 5-µl donor and acceptor vesicles in a final volume of 100 µl for 1 h at 37°C. The reaction was quenched by adding 400 µl of ice-cold 15:35 buffer (without BSA). The negatively charged donor vesicles were removed from the reaction mixture by adsorption onto DEAE cellulose (Whatman DE-52). The supernatant (containing the acceptor vesicles) was collected after low-speed centrifugation (13,000 rpm × 10 min) and recentrifuged (13,000 rpm × 5 min) to ensure total removal of the DEAE cellulose before scintillation counting. The ratio of [3H]glycerol trioleate to [14C]dipalmitoyl phosphatidylcholine was determined, and the percentage of lipid transfer was calculated from the increase in this ratio.Measurement of lipid and lipoprotein secretion. Colonic and jejunal explants were cultured (48 h) in serum-free Leibovitz L-15 medium containing a final amount of nonlabeled oleic acid (1 µmol/ml) with 0.3 µCi of [14C]oleic acid attached to albumin (sp. act. 53.9 mCi/mmol; Amersham) as reported earlier (22-24, 26-28, 32, 33, 44). The determination of individual lipid classes and lipoproteins in media by one-dimensional thin-layer chromatography and ultracentrifugation, respectively, was carried out according to the techniques described previously in detail (22-24, 26-28, 32, 33).
Labeling, immunoprecipitation, and electrophoresis of apolipoproteins. After a 48-h incubation period with [35S]methionine (100 µCi/ml) and unlabeled oleic acid (1 µmol/ml), intestinal tissues were treated for apo B immunoprecipitation, as described previously (22-24, 26, 27, 32). Apo B samples were resolved by 5% SDS-polyacrylamide gel electrophoresis, and the radioactivity in apo B was quantitated as described previously (22-24, 26, 27, 32) after the appropriate products had been excised from the gels.
Western Blots
To assess the presence of MTP and evaluate its mass, intestinal tissue was homogenized and adequately prepared for Western blotting as described previously (2). Proteins were denatured in sample buffer containing SDS andStatistical Analysis
All values are expressed as means ± SE. Mean differences between groups were calculated by unpaired Student's t-tests. ![]() |
RESULTS |
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Localization of MTP and PDI by Immunofluorescent Studies
To examine the expression of these individual proteins in the different regions of the gut and determine their distribution along the crypt-villus axis, indirect immunofluorescence was carried out. Figures 1 and 2 illustrate the localization of MTP along the crypt-villus axis in the human fetal jejunum and colon, respectively. Immunofluorescence staining was almost entirely observed in the columnar epithelial cells of both segments. MTP staining was detected as early as the 13th wk of gestation in all gut segments, including the duodenum and ileum (not shown). Cytoplasmic immunofluorescence staining was visualized in all absorptive cells located in the crypt-villus axis with no specific compartmentalization. Figure 3 illustrates the distribution pattern of PDI. Cytoplasmic immunofluorescence staining was observed in columnar epithelial cells in all gut segments as early as the 13th wk of gestation. PDI staining was detected in crypt as well as villus epithelial cells. In all intestinal segments, goblet cells appeared to be negative for MTP and PDI signals. Taking into account the qualitative nature of this technique, it was not possible to speculate on quantitative aspects relative to MTP and PDI signals.
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MTP Activity Along the Intestine
Triacylglycerol transfer activity was measured in different segments of the intestine in the linear range of the assay. MTP activity was estimated by monitoring the transfer of triacylglycerol from donor to acceptor vesicles in the presence or absence of MTP extracts prepared from jejunal, ileal, and colonic explants. MTP activity was detected in all of the intestinal regions, including the colonic segments (Fig. 4A). However, a descending gradient was apparent from the proximal small intestine to the distal colon, as shown in Fig. 4A. The calculated data disclosed that the MTP activity of the ileum, proximal colon, and distal colon represented 61.1, 8.4, and 7.5% of jejunal samples.
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Detection of MTP and PDI Proteins by Immunoblotting
Samples of the different intestinal regions containing equal amounts of protein were electrophoresed on an SDS-polyacrylamide gel. An immunoblot of these samples showed immunoreactive bands corresponding to the large subunit of MTP and to the small subunit of PDI (Fig. 4B). Consistent with activity measurements, Western blot analyses showed that the ileum, proximal colon, and distal colon contained 75, 25, and 15%, respectively, of the amount of the large subunit of MTP measured in the jejunum. In contrast, densitometric estimation of the amount of PDI visualized on the immunoblot revealed that the distal colon contained more protein than the small intestine segments.MTP Activity and Mass in Developing Intestine
Our previous studies documented a gradual increase in the synthesis of triglycerides and chylomicrons as a function of gestational age (44). In the present investigation, we explored the maturation aspect of MTP activity in the explants of the human small and large intestine between 15 and 22 wk of gestation. Only a trend toward increase was noted in the jejunal explants at the period of 20-22 wk of gestation (Fig. 5). On the other hand, no marked modification was apparent in MTP activity and mass in colonic explants with fetal age.
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Studies on MTP in Intestinal Organ Culture
In an attempt to evaluate the effect of fatty acids on MTP activity and mass, jejunal and colonic explants were cultured in serum-free Leibovitz L-15 medium supplemented with oleic acid. No marked changes were noted in MTP levels following the addition of oleic acid (Fig. 6). Furthermore, to determine whether alterations occur in MTP activity and mass as a function of incubation periods, jejunal explants were cultured at 0, 4, 8, and 24 h. As illustrated in Fig. 7, MTP peaked at 4 h and decreased progressively at 8 and 24 h.
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Studies on Lipids, Lipoproteins, and Apolipoproteins in Intestinal Organ Culture
To characterize the lipoprotein particles secreted by jejunal and colonic explants, the cultures were supplemented with 1 µmol/ml oleic acid. Since the output rate of triacylglycerol by the jejunal explants is relatively low and typical of cell culture, the explants were cultured for a 48-h incubation period. Then the conditioned media were collected, lipids were determined, apo B was analyzed, and lipoproteins were isolated by sequential ultracentrifugation. Colonic explants secreted ~20% of total lipids exported by the jejunum (Fig. 8). Under our experimental conditions, the major lipid classes esterified by the colon and transported into the medium never exceeded 30% of the jejunum's capacity. Similarly, the production of apo B by colonic explants was 30-40% of the level of jejunal explants. In association with the lower ability to transport lipids, apo B, and lipoproteins, colonic explants displayed limited triacylglycerol transfer capabilities. Both the activity and protein mass of MTP were relatively poorly expressed in colonic explants.
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DISCUSSION |
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We previously reported that triglyceride-rich particles are generated by the absorptive cells of the fetal intestine (26-28, 32, 33, 44). Furthermore, our recent studies have established that normal human crypt cells are able to express apolipoproteins and export lipoproteins, which suggests that the lipoprotein assembly process is not restricted to mature villus enterocytes (21). An important prediction arising from our reports was that MTP should be expressed in the developing gut. The present investigation provides support for this hypothesis. In the current work, a series of immunofluorescent findings have documented the presence of MTP in both villus and crypt epithelial cells of the small intestine and colon. PDI, necessary to maintain the catalytically active structure of MTP, was also identified in the same intestinal regions. Biochemical manipulations revealed substantial MTP activity, which appeared as a decreasing gradient from the proximal small intestine to the colon. Jejunal explants in culture did not show changes in MTP mass and activity following the addition of oleic acid, whereas a trend toward increase was noted over the fetal periods studied and in response to a 4-h incubation period. Importantly, colonic explants exhibited low expression of MTP. These observations combined with our previous reports demonstrate the small intestine's ability to synthesize and secrete apo B-containing lipoproteins during development. Also, the colon's limited capacity for lipid transport, in sharp contrast to the jejunum's efficiency, was associated with the reduced MTP protein amount and activity.
Assembly of triglyceride-rich lipoproteins involves a complex process in which apo B is packaged with lipids from the hydrophobic core and polar surface (9, 40, 41). Lipoprotein formation is initiated as apo B is translocated into the lumen of the endoplasmic reticulum (9, 40, 41). MTP appears to be essential to this process to form stable nascent apo B-containing lipoprotein particles in the endoplasmic reticulum, which otherwise would be degraded. The current experiments demonstrate that fetuses have the machinery in place that is necessary to assemble lipoproteins. In fact, our results indicate that MTP is already present at the gestational period to direct apo B into the lipoprotein assembly and secretion pathway. Notably, its functioning may be useful for 1) the absorption of lipids and fat-soluble compounds contained in the amniotic fluid and meconium (3); 2) fatty acids transported from mother to fetus (14); and/or 3) the preparation for postnatal life characterized by a high fat intake.
Our recent work has demonstrated the potential of the colon to absorb nutrients, including fat, at precocious stages of development (1, 22), which correlates with the formation of villi similar to those in the small intestine and the appearance of brush-border hydrolytic activities. Despite the capability of the fetal large bowel to esterify lipids and synthesize apolipoproteins such as apo B-48 and apo B-100, a striking failure in lipoprotein assembly and secretion was evidenced compared with the jejunum originating from the same fetuses (22). The defect was not localized at the level of apo B mRNA editing, since this unique posttranscriptional event producing apo B-48 via an alteration of the RNA sequence was found to be fully normal in the colon (22). The possible compounding factor leading to the impairment of lipoprotein delivery by the colon could be attributed to an abnormally low level of MTP, as demonstrated by the present data. This is strongly supported by the various roles of MTP: 1) affected individuals with abetalipoproteinemia exhibit an absolute requirement for MTP for apo B-containing lipoprotein production (2, 20, 49, 51); 2) the expression of MTP in HeLa cells, a non-lipoprotein-producing cell line, was sufficient to reconstitute the efficient assembly of apo B-53 and lipid into a macromolecular lipoprotein particle that was secreted from the cell (13); and 3) the inhibition of MTP activity in Hep G2 cells, a human liver-derived cell line that secretes apo B-containing lipoproteins, impaired lipid transport in a concentration-dependent manner (5, 16, 46). Since MTP and apo B interact during lipoprotein assembly (5, 46, 52), the reduction in the amount of MTP in colonic explants might have affected lipoprotein production. Additional work is obviously necessary to delineate whether MTP continues to be somewhat active in the mature colon or disappears at birth, thereby explaining the failure of the adult human colon to efficiently transport lipids.
The influence of oleic acid on the activity and mass of MTP was evaluated in cultured jejunal explants. Oleic acid supplements to culture media had no effect on MTP levels. These data are in agreement with those of Mathur et al. (34), who reported that, in Caco-2 cells, neither MTP activity nor MTP mass changed in response to lipid mediators known to stimulate apo B secretion. Similarly, the addition of fatty acids to Hep G2 cells did not alter MTP mRNA levels (31). The treatment of hamsters with high fat diets also resulted in a moderate increase in intestinal MTP gene expression (30). Since a hormonal regulation of apolipoproteins and lipoproteins was shown in jejunal and colonic explants (1, 26-28, 32, 33, 44), further work is needed to explore the regulation of MTP by hydrocortisone, epidermal growth factor, and insulin.
PDI is a ubiquitous protein that has multiple functions within the
lumen of the endoplasmic reticulum (12). It catalyzes the
formation of disulfide bonds in newly synthesized proteins (11), thus promoting the proper folding of
disulfide-bonded proteins (6). It also functions as the
-subunit of prolyl 4-hydroxylase, which generates 4-hydroxyproline
that in turn contributes to the stability of the collagen triple
helical structure (17). Other suggested functions of PDI
include chaperone and binding activities. Indeed, PDI serves as a
chaperone-like protein that nonspecifically binds peptides in the
endoplasmic reticulum (19, 37-39) and can act as a
major cellular thyroid hormone binding protein (7), a
developmentally regulated retinal protein termed r-cognin
(18), and dehydroascorbate reductase (47). As
illustrated by the present findings, our immunofluorescence studies
could not discriminate between the distribution patterns of MTP and PDI. As a subunit in the lipid transfer protein complex, it probably colocalizes with the 88-kDa MTP in the enterocyte during development to
confer lipid transfer activity. PDI is probably necessary for normal
cell functioning, whereas MTP may only be required for those cells that
transport triacylglycerol.
In conclusion, our immunofluorescent observations illustrate the distribution of MTP and PDI in the small and large intestine. Our systematic investigation has demonstrated for the first time that MTP and PDI are distributed along the entire length of the gut. The restricted pattern of MTP activity and protein in the colon is consistent with its reduced lipoprotein secretion capacity. Thus the present studies provide in vitro support for the hypothesis of apo B core lipidation, demonstrating the ability of MTP to modulate the assembly and secretion of triglyceride-rich lipoproteins. When lipid addition at the site of apo B assembly is reduced through low MTP activity, as observed in colonic specimens, a lower yield of apo B-containing lipoproteins is noticeable.
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ACKNOWLEDGEMENTS |
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We thank Drs. J. R. Wetterau and H. Jamil for the polyclonal antibodies. We also acknowledge the expert secretarial assistance of D. St-Cyr Huot. We thank Lina Corriveau and Louise Thibault for technical assistance and Drs. C. Poulin and F. Jacot of the Département de la Santé Communautaire for their excellent cooperation in providing tissue specimens for the study.
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FOOTNOTES |
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This study was supported by a research grant from the Medical Research Council (MRC) of Canada (MT-10583 and MGC-15186) and the Canadian Heart Association. E. Levy is a member of the MRC Research Group in the Functional Development and Physiology of the Digestive Tract.
Address for reprint requests and other correspondence: E. Levy, Gastroenterology Unit, Hôpital Sainte-Justine, 3175 Côte Ste-Catherine Road, Montréal, PQ, Canada H3T 1C5 (E-mail: levye{at}justine.umontreal.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 7 July 2000; accepted in final form 11 October 2000.
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