Departments of 1Internal Medicine and 2Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110
Submitted 5 May 2003 ; accepted in final form 9 June 2003
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ABSTRACT |
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chylomicrons; very low-density lipoproteins; apolipoprotein B-100; apolipoprotein B-48
Triglyceride-rich lipoprotein secretion in mammalian enterocytes demonstrates important differences from hepatocytes. Specifically, enterocytes manifest the unique ability to synthesize and secrete chylomicrons. In addition, mammalian enterocytes generate a truncated form of apoB (apoB-48) through posttranscriptional C to U RNA editing of the nuclear transcript that introduces a translational stop codon, resulting in a protein encoding the amino terminal 48% of apoB-100 (1). By contrast, human hepatocytes synthesize and secrete exclusively the protein product of the unedited mRNA, apoB-100. Exceptions to this organ-specific partitioning of apoB production include mouse and rat hepatocytes that express apobec-1 (the catalytic deaminase) and secrete both apoB-100 and apoB-48 (16). In addition, enterocytes from different mammalian species, including humans, contain varying amounts of unedited apoB mRNA (10%) and indeed synthesize apoB-100 (20). Furthermore, intestinal apoB mRNA editing is physiologically regulated through a variety of developmental, hormonal, and metabolic cues, suggesting an intrinsic functional advantage associated with the ability to modulate the proportions of apoB isoforms (5, 19, 28, 31, 33). Nonetheless, there has been little in vivo evaluation of the functional consequences of apoB-100 vs. apoB-48 isoform production in the context of enterocyte lipoprotein assembly and secretion.
To address the role of apoB-100 vs. apoB-48 in murine lipoprotein metabolism, we (17) and others (26, 27) undertook gene targeting of apobec-1 to eliminate C-to-U editing of apoB RNA. The functional outcome was the same in all three reports, namely, lines of mice that appeared healthy and fertile and that grew normally without an obvious difference in weight gain or in total serum cholesterol and triglyceride concentrations while consuming different diets (17, 26, 27). However, the possibility of a subtle phenotype involving intestinal lipid packaging and secretion in apobec-1/ mice remained open. Accordingly, we undertook to resolve the question of whether intestinal apoB-100 and apoB-48 are regulated through equivalent pathways involving posttranslational degradation and to refine the analysis of the functional impact of intestinal apoB-100 expression on chylomicron assembly and secretion.
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MATERIALS AND METHODS |
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Animals and feeding schedules. Apobec-1/ (knockout) mice were previously generated (17) in a mixed 129/sv background and backcrossed >12 generations onto a C57BL/6 background. Male C57BL/6 wild-type mice were purchased from Jackson Laboratories (Bar Harbor, ME). Wild-type and apobec-1/ mice were maintained on a 12:12-h light-dark cycle, in a full-barrier facility. Food intake and fecal collections were carried out where indicated by using groups of animals maintained in metabolic cages. Where indicated, 12- to 16-wk-old male mice were fed ad libitum, either a chow diet (Picolab Rodent Diet 20; fat content 4.5% wt/wt, comprising 11.9% calories) or a Western diet (cat. no. TD88137, Harlan-Teklad; fat content 21.2% wt/wt, comprising 42% of calories) for 2 wk before the study. Unless indicated otherwise, animals were fasted for 3 h before study.
Enterocyte isolation. Mice were anesthetized by using Metofane, and the small intestine was immediately removed. Attempts to isolate enterocytes from intestinal loops (7) gave inconsistent results with respect to the integrity of apoB isoforms over a 2-h radiolabeling period (data not shown). Accordingly, we adapted an enterocyte isolation protocol for the use of small, effaced sections of small intestine. The intestine was opened longitudinally, and a 30-cm region from the distal duodenum to proximal ileum was isolated, sliced into 45 pieces each 56 cm in length in that the mucosa was completely effaced. Intestinal pieces were transferred to plastic tubes and washed quickly in 50 ml buffer A (HBSS containing 1% FCS) with gentle shaking for 10 s each for a total of five washes followed by one wash in 50 ml buffer B (Ca2+- and Mg2+-free HBSS supplemented with 2% glucose and 2% BSA). After these washes at room temperature, the intestinal pieces were incubated in 25 ml prewarmed (37°C) enterocyte isolation buffer (Ca2+- and Mg2+-free HBSS supplemented with 0.5 mM DTT and 1.5 mM EDTA) at 37°C for 15 min with shaking (100 rpm/min). The cells were collected and pelleted at 800 rpm for 5 min and resuspended in 10 ml buffer B and held on ice. The intestinal pieces were then reincubated in warm isolation buffer for a second incubation of 15 min with shaking. Cells collected from the first and second isolation were pooled and resuspended in methionine- and cysteine-free DMEM (pregassed with 95% O2-5% CO2) for the experiments detailed below. Yields of enterocytes averaged 3 x 107 per animal. The volume of media (typically 57 ml) was adjusted to yield enterocyte concentrations of 1 x 107 per ml. The viability of isolated enterocytes, assessed by Trypan blue exclusion, was routinely >90%. In addition, radiolabel incorporation into TCA insoluble counts per microgram of protein was linear for 3 h (data not shown).
Pulse chase and radiolabeling experiments. Enterocytes (107/incubation) were pulse labeled for 30 min with 250 µCi/ml 35[S]protein labeling mix and chased for 120 min in DMEM supplemented with 10 mM methionine and 5 mM cysteine. A mixed micellar lipid solution [final concentrations (in mM): 0.4 sodium taurocholate, 0.54 sodium taurodeoxycholate, 0.3 phosphatidycholine, 0.45 oleic acid, 0.26 monoolein] was added where indicated to both pulse and chase media in certain experiments. Cells and media were collected by centrifugation at 2,000 rpm for 10 min. Cells were lysed in 500 µl lysis buffer (50 mM Tris, pH 7.4, and 1% Triton X-100, containing protease inhibitor cocktail) and homogenized by repeated passage through a 30-gauge needle. After lysis (validated by microscopic examination), the material was centrifuged at 14,000 rpm in a microcentrifuge for 5 min at 4°C, and the supernatant was used immediately for further study or stored at 80°C. Cell lysates and media were used for immunoprecipitation as described in Immunoprecipitation of apoB and apo A-IV. For the determination of apo synthesis rates, isolated enterocytes were radiolabeled for 15 min with 250 µCi/ml 35[S]protein labeling mix and cell lysates were prepared as described above. Aliquots of lysate were used for immunoprecipitation of apoB and apo A-IV, and for quantitatation of protein concentration and TCA insoluble radiolabel incorporation.
Immunoprecipitation of apoB and apo A-IV. Immunoprecipitation buffer (5x) was added to both lysates and media to a final concentration of (in mM) 150 NaCl, 5 EDTA, 50 Tris, 20 methionine, and 1% Triton X-100, 1 mg/ml BSA, and 0.02% Na2N3. Saturating quantities of anti-apoB or anti-apo A-IV antisera were added (validated as described below, and the solution was incubated at 4°C for 2 h on a rotating platform. Protein A-agarose beads were then added, followed by incubation at 4°C overnight on a rotating platform. The slurry was washed three times in 1 ml RIPA buffer [50 mM Tris (pH 7.4) 0.65 M NaCl, 20 mM EDTA, 1% sodium deoxycholate, 0.1% SDS, and 1% Triton X-100] and twice in water, each time followed by brief centrifugation to pellet the beads. The final pellet was boiled for 5 min in SDS sample buffer and analyzed by 415% SDS-PAGE and fluorography. Quantitation was conducted by the use of a PhosphorImager (model SI; Molecular Dynamics, Sunnydale, CA) and ImageQuant Software and the data normalized to total TCA precipitable radioactivity. Supernatant from the first immunoprecipitation was retained and subjected to reimmunoprecipitation as described above with a further aliquot of the original anti-serum to ensure that no additional material remained.
Lipoprotein fractionation by fast performance liquid chromatography. Isolated enterocytes (107/ml) were continuously labeled with 250 µCi/ml 35[S]protein labeling mix for 120 min. Media were collected, concentrated to a volume of 1 ml with a 10 K normal molecular weight limit centrifugal filter device at 4°C, and used for the isolation of lipoproteins by fast performance liquid chromatography (FPLC) with two Superose 6 columns connected in series (Pharmacia Biotech). For each analysis, 1 ml of concentrated media were applied and 40 x 0.8-ml fractions were collected on ice. The elution profiles were standardized by using human and mouse serum lipoproteins. ApoB was immunoprecipitated from pooled fractions (indicated in the relevant figure legend) and resolved by 415% SDS-PAGE. Quantitation was conducted by PhosphorImager.
Determination of intestinal triglyceride secretion in vivo. After a 16-h fast, wild-type and apobec-1/ mice were anesthetized, weighed, and injected intravenously with 20 mg (500 mg/kg body wt) Triton WR1399 (Tyloxapol; Sigma) in 100 µl saline. Immediately after tyloxapol administration, the mice received an intragastric bolus of 1 ml 20% Intralipid. Blood was collected at 0, 1, 2, 3, and 4 h and subjected to centrifugation at 4,000 rpm for 20 min, and total triglyceride content was measured enzymatically by using a Wako Chemicals L-Type TG H kit.
Electron microscopy. Pooled aliquots of sera (200 µl) from the 4-h time point (see Determination of intestinal triglyceride secretion in vivo) were layered under 600 µl 0.15 M NaCl and subject to centrifugation at 50,000 rpm for 30 min in the MLA-130 rotor in a table-top ultracentrifuge (Beckman Instruments). Chylomicrons were removed and subjected to negative staining electron microscopy by using aqueous uranyl acetate and viewed under a Zeiss 902 electron microscope. In separate experiments, isolated enterocytes were prepared from either chow- or fat-fed animals and incubated in DMEM with micelles for 120 min, and the media were collected. The density was adjusted to 1.21 g/ml by adding solid NaBr. Lipoproteins were prepared by ultracentrifugation at 100,000 rpm for 4 h at 16°C (Beckman MLA-130 rotor), dialyzed against 150 mM NaCl, 0.02% sodium azide, and 0.004 mM EDTA overnight. After centrifugal concentration with the use of a 10 K normal molecular weight limit centrifugal filter, lipoproteins were subjected to negative staining (as in Electron microscopy).
All procedures were conducted according to ethical guidelines approved by the Institutional Animal Care and Use Committee of Washington University School of Medicine.
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RESULTS |
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The optimized protocol for isolated enterocyte preparation permitted robust detection of both apoB-48 and apoB-100 and demonstrated a 1.7- to 2-fold increase in de novo synthesis of both isoforms in response to high fat intake (Fig. 1, C and D). Apobec-1/ mice demonstrated only apoB-100 in intestinal lysates, with increased synthesis after high fat consumption (Fig. 1, C and D). Inclusion of mixed micelles during the radiolabeling period in either chow or high-fat-diet-fed animals yielded similar initial synthesis rates to incubations in unsupplemented media (Fig. 1D). These findings suggest that there is comparable induction of intestinal apoB-100 and B-48 synthesis after intake of a high-fat diet with or without micellar lipid in the labeling medium.
Subtle defect in intestinal triglyceride secretion rate in apobec-1/ mice. Chow-fed wild-type and apobec-1/ mice were fasted overnight and challenged with an oral lipid bolus after tyloxapol injection. Plasma triglyceride accumulation was significantly greater in wild-type animals at 2, 3, and 4 h (Fig. 2), suggesting that there may be differences in the rate of intestinal triglyceride secretion among the different genotypes. Although periods longer than 4 h were not examined, we predict that intestinal triglyceride secretion is eventually complete, because there were no residual accumulation within enterocytes (Fig. 1B) and no difference in fecal fat content (data not shown). Accordingly, further analysis focused on the short-term regulation of apoB synthesis and secretion, as detailed below.
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Secretion of apoB isoforms from primary murine enterocytes is influenced by dietary fat intake. ApoB-48 secretion was detectable within 30 min of chase in chow-fed wild-type animals (Fig. 3A), and by 120 min of chase, 10% of the newly synthesized apoB-48 was recovered in the media (Fig. 3, A and B). After intake of a high-fat diet, apoB-48 was detectable in the media of enterocytes from wild-type animals by 15 min of chase, and secretion increased to
20% of the newly synthesized material (Fig. 3, A and B). By contrast, secretion of apoB-100 was barely detectable in chow-fed apobec-1/ mice, with <3% of the newly synthesized material recovered at 120 min of chase (Fig. 3, C and D). With high fat intake, the secretion of apoB-100 from enterocytes of apobec-1/ mice increased to
7% of newly synthesized material recovered at 120 min of chase (Fig. 3, C and D). These data demonstrate that the initial rates (0120 min) of apoB secretion, as a fraction of the pool of newly synthesized intestinal apoB, are greater for apoB-48 (wild-type) than apoB-100 (apobec-1/ mice). The results further suggest a plausible explanation for the reduced rate of intestinal triglyceride secretion in apobec-1/ mice, namely the corresponding reduction in apoB-100 secretion rates compared with that of apoB-48.
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The cumulative recovery of immunoprecipitable apoB from cell lysates (Fig. 3, A and C) permitted a determination of the degradation of intestinal apoB-100 and apoB-48. These findings demonstrate that 70% of the apoB-48 is recovered at 120 min of chase in isolated enterocytes from chow-fed wild-type animals, whereas
85% was recovered in enterocytes from animals consuming a high-fat diet. Comparable amounts of apoB-48 were retained at 120 min of chase in isolated enterocytes from both chow-fed and high-fat diet groups (
6070%, Fig. 3B), suggesting that the difference in recovery of apoB-48 with high-fat feeding is accounted for by enhanced secretion rather than altered degradation. By contrast, only
37% of the apoB-100 was recovered in chow-fed apobec-1/ mice, increasing to
65% in animals consuming a high-fat diet (Fig. 3D). This was accounted for by reduced recovery of intracellular apoB-100 in chow-fed apobec-1/ mice (
34% vs.
58%; Fig. 3D), suggesting that sustained high fat intake reduces intracellular apoB-100 degradation. Further studies were conducted to examine the role of micellar lipid.
Inclusion of micellar lipid preferentially increases secretion and decreases degradation of apoB. Inclusion of micellar lipid in the incubation medium increased secretion of apoB-48 from chow-fed wild-type mice to 25% at 120 min (Fig. 4, A and B), contrasted with 10% in unsupplemented media (Fig. 3B). Intake of a high-fat diet and inclusion of micelles, however, did not further enhance the secretion of apoB-48 above that observed in chow-fed wild-type animals (
25%, Fig. 4B). Thus despite a twofold increase in the initial synthesis rates of apoB-48 (Fig. 1A), there is an upper limit of secretion of apoB-48 (
25% at 2 h). Nevertheless, total recovery of apoB-48 increased to
81 and 94% in chow and high fat fed wild-type mice (Fig. 4B), implying that there is only limited degradation of the retained apoB-48 in the presence of micellar lipid.
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Micellar supplementation increased the secretion of apoB-100 from chow-fed apobec-1/ mice to 15% at 120 min (Fig. 4, C and D), contrasted with <3% in the absence of micelles (Fig. 3D). In addition, intake of a high-fat diet and inclusion of micelles in the incubation together increased secretion of apoB-100 approximately ninefold over chow-fed apobec-1/ mice, resulting in secretion rates of
26% at 120 min, essentially indistinguishable from the secretion rates of apoB-48 observed in wild-type mice (Fig. 4, C and D). Total recovery of apoB-100 also increased with the inclusion of micellar lipid,
7480% of the starting material being recovered (Fig. 4D).
These findings, considered together with the data presented in Fig. 3, suggest that lipid availability increases the secretion efficiency of both apoB isoforms but particularly apoB-100 as evidenced by the findings in apobec-1/ mice. Thus intrinsic differences in apoB-48 vs. apoB-100 secretion in the setting of limited lipid availability may contribute to the diminished secretion rates of intestinal triglyceride noted above.
Synthesis, content, secretion, and degradation of apo A-IV is identical in the setting of intestinal lipoprotein formation driven by apoB-48 or apoB-100. We undertook analysis of apo A-IV gene expression, another prototype chylomicron-associated apoprotein whose expression has been demonstrated to modulate intestinal triglyceride secretion (25, 30). As illustrated in Fig. 5A, synthesis of apo A-IV was increased in response to fat feeding with indistinguishable responses in wild-type and apobec-1/ mice. In addition, intracellular apo A-IV mass increased to a similar extent in these two groups in response to high fat intake (Fig. 5B). The kinetics of apo A-IV secretion was also examined, the results indicating an identical time course in both genotypes with 25% apo A-IV secreted into the media by 2 h of chase (Fig. 5, C and D) with either chow or high fat intake. Finally, the recovery of apo A-IV was essentially 100% (indicating no degradation) in both genotypes and in all settings of intestinal lipid availability (Fig. 5, C and D). These data collectively demonstrate that neither apo A-IV gene expression nor its secretion is influenced by the apoB isoform composition of intestinal lipoproteins and is thus unlikely to contribute to the defect noted in triglyceride secretion in apobec-1/ mice.
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Intestinal lipoprotein distribution reveals distinct populations containing apoB-100 and apoB-48. Media from radiolabeled enterocytes were subjected to FPLC separation and fractions analyzed by immunoprecipitation to determine the distribution of nascent apoB-100 and apoB-48. Intestinal apoB-48 secreted from isolated wild-type enterocytes distributed predominantly into small lipoproteins in the range of HDL size particles with a second peak noted in the range of very low-density lipoproteins (Fig. 6A). After intake of a high-fat diet, there was a subtle shift with enrichment of apoB-48 noted in the larger lipoprotein particles (Fig. 6A). It bears emphasis, however, that apoB-48 was found in a continuous size distribution from HDL through to large VLDL. By contrast, analysis of intestinal lipoproteins from apobec-1/ mice revealed that apoB-100 was confined to the larger particles, in both chow-fed and high-fat-fed animals (Fig. 6B). There were no apoB-100-containing lipoprotein particles identified in the HDL size range. The small amounts of apoB-100 secreted from wild-type mice also distributed into large particles (Fig. 6A, fraction 2).
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Secretion of larger lipoprotein particles with apoB-100. In a separate series of experiments, media were collected from isolated enterocytes and lipoproteins (density <1.21 g/ml) prepared by ultracentrifugation. Negative stain examination revealed that the size distribution shifted into significantly larger particles in lipoproteins recovered from the apobec-1/ mice (Fig. 7 AD). To extend these findings to an in vivo setting, chow-fed mice were administered an oral triglyceride bolus after intravenous tyloxapol injection. Serum chylomicrons were prepared by ultracentrifugation 4 h after oral triglyceride administration. Negative stain electron microscopy reveals that chylomicron particles from apobec-1/ mice were significantly larger than chylomicrons from wild-type controls (Fig. 7, EH), consistent with the predictions above that apoB-100 secretion is associated with larger particles than apoB-48.
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DISCUSSION |
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In regard to the first question, the present findings suggest that apobec-1/ mice manifest a subtle defect in the initial rates at which triglyceride is secreted from enterocytes after an oral lipid challenge. The most likely explanation for this defect is that the secretion of apoB-100 is intrinsically less efficient than apoB-48. Nevertheless, despite this subtle defect in the initial rates of intestinal triglyceride secretion, the data reveal similar weight gain and food intake in both genotypes and comparable fecal fat and residual enterocyte triglyceride content. These findings considered together suggest that intestinal triglyceride secretion in apobec-1/ mice most plausibly occurs over a longer time frame than in wild-type animals. In addition, the findings in both serum chylomicrons and nascent lipoproteins secreted from isolated enterocytes reveals the presence of larger particles in association with apoB-100. These findings strongly imply that apobec-1/ mice, in response to an oral lipid challenge, secrete fewer but larger lipoprotein particles.
The present findings should be interpreted in the context of earlier reports of the lipoprotein phenotype of mice expressing an apoB-100-only profile. Specifically, one of the original reports of the apobec-1/ phenotype demonstrated no differences in chylomicron particle size from intestinal lymphatics of apobec-1/ and wild-type control mice, on either a low or high-fat diet and no difference in vitamin A delivery into serum chylomicrons (26). Indeed the conclusion of this, as well as our own initial characterization of apobec-1/ mice, was that there was no obvious impairment of intestinal lipid absorption (17, 26). Young and colleagues (13) reached similar conclusions in a comparison of apoB-48-only and apoB-100-only mice, demonstrating comparable -tocopherol levels, and no evidence of intestinal lipid accumulation on either a chow or high-fat diet. Thus the possibility of a subtle defect in intestinal triglyceride secretion was not explored in these earlier reports.
This possibility was addressed in a more recent report (18). Specifically, Higgins and colleagues (18) demonstrated that apobec-1/ mice demonstrate impaired intestinal triglyceride secretion with a threefold increase in residual enterocyte triglyceride accumulation in animals fed a low-fat diet. In addition, there was a paradoxical and unexplained increase in newly synthesized enterocyte apoB secretion from fasted apobec-1/ mice compared with wild-type controls (18), findings contrary to those reported in the present study. The studies of Higgins and colleagues (18) were performed in mice whose genetic background was not defined, raising the possibility that other confounding variables might contribute to the phenotype demonstrated in their report. Accordingly, the present findings, considered in the context of the original reports of the apobec-1/ phenotype, emphasize the importance of examining disparate elements of murine lipoprotein metabolism, including intestinal cholesterol absorption, and intestinal lipoprotein assembly and secretion, in a defined inbred genetic background, for example C57BL/6 (34). This approach has now facilitated characterization of a subtle but reproducible phenotype in relationship to intestinal assembly and secretion of apoB-100 not apparent in earlier studies. The present studies have also established that enterocytes from animals expressing either apoB-100 or apoB-48 show indistinguishable patterns of apo A-IV gene expression, including synthesis, secretion, content, and essentially quantitative recovery. Thus induction of apo A-IV synthesis and secretion in apobec-1/ mice does not alone compensate for the subtle defect noted in triglyceride secretion.
A second objective of the present study was to establish the role of posttranslational degradation of distinct isoforms of intestinal apoB in relationship to lipid availability. Study of intestinal lipoprotein assembly and in particular, detailed analysis of the synthesis, intracellular degradation, and secretion kinetics of apoB has been hindered by the lack of a suitable model for short-term enterocyte culture. To place this in context, recent studies (23) of apoB degradation in Caco-2 cells, a cell culture model widely used as a surrogate for intestinal lipoprotein assembly and secretion, reported that neither apoB-100 nor apoB-48 was degraded, regardless of lipid availability. Because there are now an increasing number of mouse genetic models of altered intestinal lipid metabolism (6), one of the central objectives of this study was to establish and validate a system for the study of intestinal lipoprotein assembly and secretion in isolated murine enterocytes. The present findings establish that both apoB-100 and apoB-48 are subject to intracellular degradation, in a manner responsive to lipid availability. Furthermore, under all conditions examined, apoB-100 was more extensively degraded than apoB-48. We speculate that the intrinsic susceptibility of apoB-100 to co- or posttranslational degradation accounts for the observation that fewer lipoprotein particles are secreted from enterocytes of apobec-1/ mice compared with apoB-48 containing particles secreted from their wild-type controls. In addition, the longer time interval required for translation of each molecule of apoB-100 vs. apoB-48 would logically suggest that primordial lipoprotein formation is slower with the larger isoform.
The present report demonstrates that intestinal apoB isoform secretion is differentially regulated in response to lipid flux. Data from chow-fed animals indicated that <3% of enterocyte apoB-100 was secreted at 2 h of chase, compared with 25% in high-fat fed animals, studied in micelle-supplemented media. By contrast, apoB-48 secretion increased twofold with fat feeding, with or without micellar lipid supplementation. These findings suggest that bulk lipidation of apoB plays an important role in modulating the degradation and secretion of both isoforms, but micellar lipid plays a particularly important role in regulating apoB-100 degradation and secretion. These latter findings are reminiscent of findings in HepG2 cells in which oleate availability plays a role in regulating apoB-100 degradation at multiple steps in the secretory pathway (14). It is worthy of emphasis that the findings in relationship to apoB secretion from isolated murine enterocytes are somewhat at variance with data reported by using isolated rabbit enterocytes (7, 8, 10). Findings in this animal model suggested apoB-48 degradation was virtually eliminated with inclusion of micelles, yet despite the preservation of intracellular apoB-48, <5% was secreted at 90 min (compared to
25% from murine enterocytes in the present study). In addition, these authors noted that apoB secretion was undetectable in the absence of micellar lipid, findings divergent from the present results in which
19% of apoB-48 and 7% of apoB-100 was secreted without micellar supplementation (Fig. 3B). Finally, >90% of the apoB-48 secreted from rabbit enterocytes was associated with chylomicrons with virtually none in fractions of higher density, findings in marked contrast to the present findings in wild-type mice (Fig. 6A). Accordingly, an important conclusion to emerge from the present studies is that findings in regard to intestinal apoB-100 and apoB-48 assembly and secretion may not necessarily be extrapolated among species.
A further conclusion from the present study is that enterocytes expressing apoB-100 secrete larger lipoprotein particles than enterocytes expressing apoB-48. It should be noted that Higgins and colleagues (18) found larger serum chylomicrons accumulating in their fat-fed apobec-1/ mice compared with wild-type controls, findings confirmed in the present studies. The present studies extend these findings with the demonstration that isolated enterocytes from apobec-1/ mice secreted no HDL-ized particles containing apoB-100. By contrast, wild-type control mice secreted apoB-48-containing particles that migrated throughout the size range but predominantly into HDL and chylomicron/VLDL fractions. The observation that apoB-48 associates with HDL-sized lipoprotein particles is certainly consistent with predictions on the basis of current concepts of triglyceride-rich lipoprotein particle formation. These include a dominant role of the MTTP in lipidation of the nascent apoB polypeptide and formation of small dense particles that accumulate additional core lipid (the so-called second step) that generates particles in the VLDL/chylomicron size range (12). Intracellular lipoprotein particles containing apoB-48 have been isolated from the smooth endoplasmic reticulum of rabbit enterocytes, which migrate as small HDL-sized particles and appear to be the metabolic precursors of chylomicrons (9, 10). Accordingly, one testable hypothesis to emerge from the present studies is that the intracellular pathways for apoB-48 and apoB-100 diverge at an early stage such that a population of apoB-48-containing HDL-sized particles may enter the secretory pathway without further lipidation, whereas apoB-100-containing lipoproteins must be fully lipidated before secretion. A corollary prediction is that lipidation of apoB-100 by MTTP is less efficient than that occurring with apoB-48. These predictions will require formal examination by using subcellular fractionation and are the focus of ongoing studies.
As a final comment, the underlying importance of these divergent pathways for the regulation of apoB-100 and apoB-48 within mammalian enterocytes should be placed into physiological context. In general, 1020% apoB mRNA within adult human and murine enterocytes encodes apoB-100 and this proportion is not altered in response to lipid challenge (15, 24). However, fetal human and murine enterocytes express predominantly unedited apoB mRNA and the regulation of apoB-100 secretion in this setting has been extensively examined by using in vitro cultures (4, 21, 22). Detailed characterization of the regulation of intestinal apoB-100 secretion in this setting and its role in the developing enterohepatic circulation of sterols and other lipids thus represents an important objective with implications for nutrient delivery in the fetus. This and other issues will be the focus of future reports.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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