Intestinal lipoprotein assembly in apobec-1/ mice reveals subtle alterations in triglyceride secretion coupled with a shift to larger lipoproteins

Yan Xie,1 Fatiha Nassir,1 Jianyang Luo,1 Kimberly Buhman,1 and Nicholas O. Davidson1,2

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Mammalian enterocytes express apolipoprotein (apo)B-48, which is produced after posttranscriptional RNA editing of the nuclear apoB-100 transcript by the catalytic deaminase apobec-1. Earlier studies in apobec-1/ mice revealed an apoB-100-only lipoprotein profile but no gross defects in triglyceride absorption. However, subtle defects may have been obscured by the mixed genetic background. In addition, the intrinsic susceptibility to proteolytic degradation of intestinal apoB-100 and apoB-48 has been questioned. Accordingly, we examined triglyceride absorption, intestinal apoB expression, and lipoprotein secretion in apobec-1/ mice backcrossed into a C57BL/6 background. Inbred apobec-1/ mice absorb triglyceride normally, yet secrete triglyceride-rich lipoproteins more slowly than wild-type congenic controls. There was comparable induction of apoB synthesis in response to fat feeding in both genotypes, but apoB-100 was preferentially retained and more extensively degraded than apoB-48. By contrast, synthesis, secretion, and content of apo A-IV were indistinguishable in apobec-1/ and wild-type mice with 100% recovery, suggesting no degradation of this apoprotein in either genotype. Newly secreted lipoproteins from isolated enterocytes of wild-type mice revealed apoB-48 in both high-density lipoproteins and very low-density lipoproteins. By contrast, apobec-1/ mice secreted apoB-100-containing particles that were almost exclusively in the low and very low-density lipoproteins range with no apoB-100-containing high-density lipoproteins. These studies establish the existence of preferential degradation of intestinal apoB-100 and subtle defects in triglyceride secretion in apobec-1/ mice, coupled with a shift to the production of larger particles, findings that suggest an important divergence in intestinal lipoprotein assembly pathways with the different isoforms of apoB.

chylomicrons; very low-density lipoproteins; apolipoprotein B-100; apolipoprotein B-48


THE LAST DECADE HAS WITNESSED considerable advances in our understanding of the process of intestinal lipid absorption (11, 32). After uptake and reesterification of dietary fatty acids, triglyceride synthesis and chylomicron assembly take place through a series of events culminating in a physical interaction between microsomal triglyceride transfer protein (MTTP) as a neutral lipid donor and a large hydrophobic transport protein, apolipoprotein B (apoB) (2, 3, 36). Deletions or mutations in the structural genes encoding either of these two obligate partners lead to defects in lipoprotein assembly and triglyceride secretion (29, 35, 37). Elucidation of this phenotype has provided important insight into the mechanisms linking triglyceride flux and posttranslational stability of apoB (12). Degradation of newly synthesized apoB in HepG2 cells is mediated through proteasomal and nonproteasomal pathways and is regulated in response to lipid (i.e., oleate) supplementation (14). By contrast, studies in Caco-2 cells concluded that newly synthesized apoB was quantitatively recovered and not subject to intracellular degradation, with or without lipid supplementation (23). This divergence reveals an unresolved paradox in the posttranslational regulation of intestinal and hepatic apoB gene expression, specifically in relationship to the requirement for lipidation of apoB.

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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Materials. DMEM, methionine- and cysteine-free DMEM, fetal bovine serum, HBSS, Ca2+- and Mg2+-free HBSS, and DTT were all obtained from Life Technologies (Gaithersburg, MD). 35[S]protein labeling mix (1175Ci/mol) was purchased from New England Nuclear Life Science Products (Boston, MA). Protein A-agarose was obtained from Pierce (Rockford, IL). Complete miniprotease inhibitor cocktail tablets were purchased from Roche Diagnostics (Indianapolis, IN). Polyclonal rabbit anti-mouse apoB and anti-mouse apo A-IV antisera were generated by using purified murine LDL or recombinant murine apo A-IV, respectively, as immunogens. The specificity of antisera was confirmed by Western blot analysis. Anti-heat shock protein-40 IgG was purchased from StressGen. Centrifugal filter devices were purchased from Millipore (Bedford, MA). Sodium taurocholate was purchased from Calbiochem (San Diego, CA). The 20% Intralipid was obtained from Fresemius Kabi Clayton. Quantitation of serum triglyceride was undertaken by using an L-type TG H kit, purchased from Wako Chemicals. All other reagents were purchased from Sigma and were of the highest quality available.

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 4–5 pieces each 5–6 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 5–7 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 4–15% 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 4–15% 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.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Dietary triglyceride absorption and characterization of apoB-48 and apoB-100 synthesis in isolated primary murine enterocytes. Weight gain (Fig. 1A), food consumption and fecal fat content (data not shown) were indistinguishable in chow-fed as well as high-fat-diet-fed (data not shown) wild-type and apobec-1/ mice. Furthermore, triglyceride content of isolated enterocytes was comparable in both genotypes from both chow and high-fat-diet groups with or without a 16-h fast (Fig. 1B). These results confirm that the gross parameters of dietary triglyceride absorption are preserved in apobec-1–/– mice on a C57BL background with no apparent residual intestinal triglyceride accumulation.



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Fig. 1. Parameters of growth, intestinal triglycerides, and apolipoprotein (apo)B expression in wild-type (WT) and apobec-1/ mice. A: comparable growth curves in both genotypes from suckling to early adulthood on either chow or high-fat (HF) diet. The respective genotypes are indicated by symbols, n = 7–9 mice per group. B: comparable enterocyte triglyceride content in WT and apobec-1/ mice on either chow or HF diet, fed ad libitum or fasted overnight. C: apoB-100 and B-48 synthesis in response to HF intake. Enterocytes from WT and apobec-1/ mice were radiolabeled for 15 min with or without micellar lipid. Cellular apoB was immunoprecipitated and resolved by 4–15% SDS-PAGE. Radiolabel incorporation into apoB isoforms was determined by PhosphorImager analysis and normalized to total radiolabel incorporation. Representative images illustrate the migration of apoB isoforms. D: bar graphs depict the results from 5 individual experiments, each analyzed in duplicate. Data are expressed as percentage of PhosphorImager Units (PIU)/TCA precipitated counts (means ± SD). Bars indicates studies performed in the presence of micellar lipid (MATERIALS AND METHODS). *P < 0.05 by Student's t-test.

 

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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Fig. 2. Intestinal triglyceride secretion in WT and apobec-1/ mice. Mice of the indicated genotype were fasted overnight and administered an intravenous bolus of tyloxapol, followed by an intragastric bolus of Intralipid (MATERIALS AND METHODS). Serum triglyceride levels were determined enzymatically at the indicated times. Values are presented as means ± SD (n = 6 for WT, n = 5 for apobec-1/). *P < 0.01 by Student's t-test.

 

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 (0–120 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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Fig. 3. Intestinal apoB-48 and apoB-100 synthesis and secretion: modulation by dietary fat content. Enterocytes were isolated from WT (A and B) and apobec-1/ mice (C and D) and fed either a chow or HF diet for 2 wk before the study. Cells were pulse labeled for 30 min and chased for <=120 min. Lysates and media were collected at the indicated times. ApoB isoforms were immunoprecipitated (A and C) and quantitated by SDS-PAGE and PhosphorImager analysis. ApoB was quantitated in cell lysates and media and is expressed as a percentage of initial label incorporation at the beginning of the chase. The data are from 3–5 individual experiments, each using enterocytes pooled from 2–4 animals and are expressed as means ± SD. In some cases, the SD falls within the symbol for the means. *P < 0.05 vs. chow fed using Student's t-test.

 

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 (~60–70%, 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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Fig. 4. Intestinal apoB-48 and apoB-100 synthesis and secretion: role of micellar lipid supplementation. Enterocytes were isolated from WT (A and B) and apobec-1/ mice (C and D) fed either a chow or HF diet for 2 wk before the study. Cells were supplemented with micellar lipid (MATERIALS AND METHODS) and were pulse labeled for 30 min and chased for <=120 min. Lysates and media were collected at the indicated times. ApoB isoforms were immunoprecipitated (A and C) and quantitated by SDS-PAGE and PhosphorImager analysis. ApoB was quantitated in cell lysates and media and expressed as a percentage of initial label incorporation at the beginning of the chase. Data are from 3–5 individual experiments each using enterocytes pooled from 2–4 animals and are expressed as means ± SD. In some cases, the SD falls within the symbol for the means. *P < 0.05 vs. chow fed using Student's t-test.

 

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, ~74–80% 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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Fig. 5. Intestinal apo A-IV gene expression: response to dietary fat feeding is indistinguishable in a background of apoB-100 or apoB-48. A: HF diet increases the synthesis of apo A-IV in both WT and apobec-1/ mice. Enterocytes were radiolabeled for 15 min and apo A-IV was immunoprecipitated and quantitated after SDS-PAGE and PhosphorImager analysis. Results are expressed as percentage of PhosphorImager Units (PIU)/TCA counts (means ± SD, n = 5). B: apo A-IV mass increases in response to HF diet in both WT and apobec-1/ mice. Enterocyte lysates (50 µg protein) from chow and HF-fed animals were resolved through denaturing SDS-PAGE and were analyzed by Western blot with anti-apo A-IV antisera or anti-heat shock protein-40 as a loading control. This is representative of 3 experiments. C: secretion and recovery of enterocyte apo A-IV in chow-fed animals. Enterocytes were pulse labeled for 30 min and chased for <=120 min. Lysate and media apo A-IV were immunoprecipitated and quantitated as a percentage of the initial label incorporation at the beginning of the chase. Data are derived from 4 independent experiments and are shown as means ± SD. D: secretion and recovery of enterocyte apo A-IV in HF-fed animals. Enterocytes were pulse labeled for 30 min and chased for <=120 min. Lysate and media apo A-IV were immunoprecipitated and quantitated as a percentage of the initial labeled incorporation, assuming that radiolabeled cellular apo A-IV at the beginning of the chase represents 100%. Data were derived from 4 independent experiments and are shown as means ± SD.

 

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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Fig. 6. Intestinal lipoprotein distribution of apoB-48 is distinct from that of apoB-100. A: enterocytes were isolated from WT mice fed chow or HF diet for 2 wk and were continuously radiolabeled for 120 min; media were collected, concentrated, and separated by fast performance liquid chromatography. ApoB was immunoprecipitated from pooled fractions and subjected to SDS-PAGE and PhosphorImager analysis. The pooled fractions 1-3 represent chylomicrons (CM)/VLDL; fractions 4-6, IDL/LDL; fractions 7-9, HDL. Data are representative of 3 independent experiments (left). Relative distribution of apoB-48 is plotted (right). B: enterocytes from apobec-1/ mice fed chow or HF diets were radiolabeled and analyzed as described above. The relative distribution of apoB-100 is plotted (right). Data are representative of 3 independent experiments. IDL, intermediate density lipoprotein.

 

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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Fig. 7. Electron microscopy of intestinal lipoproteins secreted from WT and apobec-1/ mice. Media were collected from isolated enterocytes from 4–6 animals of the indicated genotype after 2 wk of HF intake (AD). Lipoproteins from the media were floated by ultracentrifugation at a density <1.21 g/ml and were subjected to negative stain and electron microscopy. Representative images (A and C; magnification, x22,000) are shown with the histogram of size distribution (200 particles counted per sample). Serum chylomicrons (density <1.006) were isolated from pooled samples taken 4 h after an oral triglyceride bolus and were subjected to negative stain electron microscopy (EH). Representative images (E and G; magnification, x14,000) are shown from5WT(E and F) and 5 apobec-1/ mice (G and H) with the histogram of size distribution (200 particles counted per sample).

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The present study was undertaken to resolve two distinct but related questions concerning intestinal lipoprotein metabolism and apoB isoform production. The first question concerned the functional role of apoB-100 vs. B-48 in the delivery of dietary triglyceride for secretion. The second and related question concerned the intrinsic susceptibility of apoB-100 and apoB-48 to intracellular degradation within small intestinal enterocytes.

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 {alpha}-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, ~10–20% 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.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This work was supported by National Institutes of Health Grants HL-38180, DK-56260, and DDRCC P30-DK-52574.


    ACKNOWLEDGMENTS
 
The authors acknowledge many useful conversations with our colleagues Libby Newberry, Susan Kennedy, and other members of the Davidson lab.


    FOOTNOTES
 

Address for reprint requests and other correspondence: N. O. Davidson, Box 8124, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110 (E-mail: nod{at}im.wustl.edu).

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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