Hypobetalipoproteinemic Mice with a Targeted Apolipoprotein (Apo) B-27.6-specifying Mutation IN VIVO EVIDENCE FOR AN IMPORTANT ROLE OF AMINO ACIDS 1254–1744 OF ApoB IN LIPID TRANSPORT AND METABOLISM OF THE ApoB-CONTAINING LIPOPROTEIN*

Carboxyl-terminal deletion of apoB-100 may impair its triglyceride (TG)-transporting capability and alter its catabolism. Here, we compare our newly generated apoB gene ( Apob )-targeted apoB-27.6-bearing mice to our previously reported apoB-38.9 mice to understand further the relationship between the size of a truncated apoB variant and its function/metabolism in vivo . The apoB-27.6-specifying mutation produces a premature stop codon six amino acids (aa) downstream of the last codon of mouse Apob exon 24 (corresponding to aa 1254 of human apoB-100). ApoB-27.6 transcripts were 3- and 5-fold more abundant than apoB wild type and apoB-38.9 transcripts in the liver. Likewise, hepatic secretion rates of apoB-27.6 were 7-fold higher than those of apoB-48 and apoB-38.9. In contrast, apoB-27.6 heterozygotes ( Apob 27.6/ (cid:1) ) had lower hepatic TG secretion rates and higher liver TG contents than both apoB-38.9 heterozygotes ( Apob 38.9/ (cid:1) ) and apoB wild type mice ( Apob (cid:1) / (cid:1) ). ApoB-27.6 was secreted by Apob 27.6/ (cid:1)

Apolipoprotein (apo) 1 B is the primary structural protein of very low density lipoproteins (VLDL) and chylomicrons. It plays a crucial role in triglyceride export by the liver and intestine and in transport of lipids in yolk sacs that is essential to normal mouse embryonic development (1,2). The full-length apoB (apoB-100) contains 4536 amino acids (3) that are predicted to conform a pentapartite structure composed of three amphipathic ␣-helical domains alternating with two ␤-strand domains (4). Carboxyl-terminal truncations of apoB-100 resulting from nonsense and frameshift mutations in the apoB gene (Apob) cause familial hypobetalipoproteinemia (FHBL) in humans, an autosomal codominant disorder characterized by low levels (Ͻ5th percentile) of plasma apoB and low density lipoprotein (LDL)-cholesterol (5,6) that is frequently associated with fatty livers (7)(8)(9)(10)(11). Numerous Apob mutations that produce truncated forms of apoB with predicted sizes ranging from apoB-2 to apoB-89 have been identified in FHBL subjects (5,6,12). ApoB-31 and larger variants of truncated apoB are detectable at low levels in plasmas of human FHBL subjects, but truncated apoB variants smaller than apoB-31, such as apoB-29 (13) and apoB-24 (14), are usually not detectable (5,6,(12)(13)(14)(15). The molecular mechanism(s) underlying these metabolic derangements is(are) still poorly understood. An elucidation of these mechanisms may lead to a better understanding of the structure-function relationship of the apoB molecule.
The production, intravascular metabolism, and clearance of lipoproteins is a complex process, and plasma levels of truncated apoB-containing lipoproteins may be low for a variety of reasons. ApoB-89 (16) apoB-75 (17) are produced at slightly lower rates than apoB-100-containing lipoproteins and are also cleared more rapidly from plasma. These "large" truncated apoB variants manifest increased affinities for the LDL receptor and are catabolized primarily in liver (16,17). Particles containing "intermediate"-sized variants of truncated apoB such as apoB-70.5 (18), apoB-54.8 (19), apoB-38.9 (20), or apoB-31 (21) are produced at lower rates than the larger forms of truncated apoB, but they are not recognized by LDL receptors (22). By analogy to apoB-48-containing lipoproteins (23), the majority of these particles probably associates with apoE in plasma and is cleared by liver via LDL receptors and the LDL receptor-related protein, but a large proportion of them is catabolized in the kidney (22,24), probably mediated by megalin (22). In contrast, the mechanisms underlying the absence of "small" truncated apoB variants (ϽapoB-31) in plasma have not been elucidated, because a suitable mouse model for such studies is not available. In fact, despite the availability of several lines of mice bearing various forms of truncated apoB (25)(26)(27)(28), apoB-38.9 (28) is the smallest variant of truncated apoB produced by those mice reported thus far.
Carboxyl-terminal truncation of apoB also impairs its triglyceride-transporting capability. We have recently demonstrated that an apoB-38.9-specifying mutation in mice reduced hepatic triglyceride secretion rates and caused fatty livers, albeit rates of apoB-38.9 secretion by the hepatocytes were not altered (28). Kim et al. (27) also reported that the particle volume of the VLDL produced by the apoB-39-only mice was reduced by 50%, compared with the VLDL produced by the apoB-48-only mice. Unlike apoB 38.9, truncated apoB variants smaller than apoB-37 are not present in VLDL and circulate at HDL densities in FHBL subjects (21,29), as do lipoproteins containing small truncated apoB variants produced by transfected hepatoma cells (30,31). Although these earlier studies suggested that the small variants of truncated apoB had a much smaller triglyceride-transporting capacity than the larger ones, the in vivo function of the small truncated apoB variant has not been adequately studied.
To understand further about the relationship between the size of a truncated apoB variant and its function/metabolism in vivo, we now report on our new apoB-27.6-bearing FHBL mouse. The availability of our previously reported apoB-38.9bearing mice (28) and of these apoB-27.6 mice has permitted the comparison of the metabolism and some of the functions of these truncated apoB variants with each other and with the normal variant apoB-48 found in wild type mice. We report on rates of embryonic production, on the transport of hepatic lipids, on the hepatic metabolism of apoB in vivo and ex vivo, and on the differential role of apoE in their clearance. Our results suggest that the peptide segment of apoB between carboxyl termini of apoB-27.6 and apoB-38.9, namely amino acids (aa) 1254 -1744, plays important roles in governing the apoB function and metabolism of the apoB-containing lipoprotein.

EXPERIMENTAL PROCEDURES
Production of ApoB-27.6 Mice-The apoB-27.6 mice were generated using a clone of homologous recombinant ES cells (clone 83) (28) bearing a targeted Apob allele in which nucleotide 5449 was deleted and a loxp-PKG-Neo-loxp cassette was inserted into exon 24 (Fig. 1A). Preparation of the targeting construct and procedures of ES cell culture and screening of homologous recombinant clones have been described (28). Briefly, a replacement-type targeting construct (Fig. 1A) was prepared using a 11.5-kb BglII-BglII genomic fragment spanning mouse Apob intron 23 through the 5Ј-end of exon 27 isolated from a BAC clone, which contains a mouse Apob insert derived from W-4 (129/SvJ) ES cell genomic DNA (clone 12339) (Genome System, St. Louis, MO). The parental ES cells were derived from 129/SvJ blastocysts (T. J. Ley, Washington University School of Medicine). After transfection of ES cells with the gel-purified targeting construct, G418-resistant clones were isolated as described previously (32) and screened by Southern blotting using a 0.3-kb HindIII-HpaI genomic fragment (Fig. 1A) as an external probe. The fidelity of the targeted mutation was confirmed by sequence analysis.
The homologous recombinant ES cells were used for micro-injection without being subjected to stable transfection with a Cre-recombinase expressing vector. C57BL/6 blastocysts were microinjected with these ES cells and implanted into pseudopregnant Swiss Webster foster females as described previously (32) to produce chimeric progeny. Male chimeras were bred with C57BL/6 females to generate heterozygous apoB-27.6 mice (apob ϩ/27.6 ). The apob ϩ/27.6 mice were intercrossed to create homozygotes (apob 27.6/27.6 ). They were also bred with apoB-38.9only (apob 38.9/38.9 ) mice (28) to generate apob 27.6/38.9 mice. All of the above offspring had a mixed genetic background with 50% C57BL/6 and 50% 129/SvJ. In addition, ApoE-null mice (29) (The Jackson Laboratory, Bar Harbor, ME) were crossbred with either apob ϩ/38.9 (33) or apob ϩ/27.6 mice. The offspring were intercrossed to produce apoE-null mice heterozygous for apoB-38.9 or apoB-27.6. The resultant mice had a mixed genetic background with 75% C57BL/6 and 25% 129/SvJ. All mice were weaned at 3 weeks of age, housed in a specific-pathogen free barrier facility with a 12-h light/dark cycle, and fed a regular mouse chow diet (Ralston Purina, St. Louis, MO).
RNA Analysis-Total RNA was isolated from mouse livers by a single-step isolation method using RNAzol B (Tel-Test, Inc.). A one-tube RT-PCR kit (Roche Biochemicals, Indianapolis, IN) was used for reverse transcriptase (RT)-PCR using primers corresponding to mouse Apob exon 22, exon 23, exon 25, various regions of intron 24, or the neomycin-resistant gene Neo r as specified in legends to Fig. 2. The RT-PCR products were gel-purified and sequenced on an ABI PRISM 377 DNA sequencer (Applied Biosystems, Foster, CA).
Northern blot analysis was used to determine the size and the levels of Apob transcripts in the livers of apoB-27.6-producing mice and their wild type littermates. Thirty micrograms of liver total RNA pooled from three mice for each apoB genotype was separated by electrophoresis on a 0.8% agarose-formaldehyde gel and transferred and immobilized onto a GeneScreen nylon membrane (PerkinElmer Life Sciences, Boston, MA). A 789-bp fragment of mouse apoB cDNA encoding amino acids 26 -289 of mouse apoB (28), which was generated by RT-PCR using an upstream primer 5Ј-TACGTGTACAACTATGAAGCT-3Ј and a downstream primer 5Ј-ACGTGGACTTGGTGCTCTC-3Ј, was radiolabeled with [␣-32 P]dCTP using a random-primer labeling kit (Roche Molecular Biochemicals Corp., Indianapolis, IN) and used to probe Apob transcripts. Northern hybridization was carried out using Rapid-hyb buffer according to the manufacturer's instruction (Amersham Biosciences, Inc., Arlington Heights, IL). The blots were also stripped and probed with a rat ␤-actin cDNA (Sigma Chemical Co., St. Louis, MO). The hybridization signals were quantified using the GS-525 phosphorimaging system (Bio-Rad, Hercules, CA). The relative apoB mRNA levels are expressed as ratios of apoB mRNA/␤-actin mRNA. Data are presented as mean Ϯ S.D. (n ϭ 3 measurements).
Western Blot Detection of ApoB and Lipoprotein Fractionation-To perform Western blot analysis, mouse plasma (2 l/well) or liver homogenates (100 g of protein/well) were subjected to electrophoresis on 3-12% gradient SDS-PAGE gels under reducing conditions and electrotransferred onto Immobilon-P (Millipore Corp., Bedford, MA). Western blot analyses were carried out using rabbit anti-mouse apoB antisera (1:10,000) or anti-GST-mouse apoB amino acids 26 -289 (28) and an ECL Western blot detection kit (Amersham Biosciences, Inc.). The ECL signals were quantified by analyzing the density of the protein bands on x-ray film using a Sigma gel computer software (SPPS Science Corp., Chicago, IL).
A fast-performance liquid chromatography (FPLC) Superose column (34) was used to assess the distribution of lipids and apoB within the lipoprotein fractions of mouse plasma. The distribution of apoB in each lipoprotein fraction was determined by Western blot analysis.
Metabolic Labeling and Pulse-chase Studies Using Mouse Hepatocytes-Hepatocytes were isolated from 10-week-old mice as described (28). Viability of the cells was about 80% as determined by Trypan exclusion. Cells were plated onto 6-well plates (0.6 ϫ 10 6 cells/well) or 100-mm dishes (7 ϫ 10 6 cells/dish) coated with poly-D-lysine (Sigma Chemical Co., St. Louis, MO) and incubated at 37°C under 5% CO 2 in 10% fetal bovine serum (FBS)/Dulbecco's modified Eagle's medium (DMEM). After 1 h of attachment, the cell monolayers were washed twice and incubated in 10% FBS/DMEM until used. All experiments involving cultured hepatocytes were commenced 7-8 h after the cells were cultured. Following this initial culture period, cells were washed three times with phosphate-buffered saline and incubated in methionine (Met)-and cysteine (Cys)-free DMEM for 30 min to deplete the cellular pool of Met and Cys. Thereafter, the medium was replaced with 1 ml of Met-and Cys-free DMEM containing 200 Ci of 35 S-Promix (530 MBq/ml, Amersham Biosciences, Inc.), an L-[ 35 S]Met and L-[ 35 S]Cys metabolic labeling solution, and cells were labeled for 3 h to determine rates of apoB secretion. At the end of this incubation period, medium was collected and spun to remove trace amounts of the cells while the cell monolayers were lysed in an immunoprecipitation buffer (IP buffer) (150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.4, 0.0625 M sucrose, 0.5% Triton X-100, and 0.5% sodium deoxycholate). A mixture of protease inhibitors was added to the media and cell lysates to a final concentration of 1ϫ according to manufacturer's instruction (Roche Molecular Biochemicals Corp., Indianapolis, IN).
Pulse-chase experiments were carried out on the apoB-27.6 heterozygous hepatocytes to compare the secretion efficiency of apoB-48 and apoB-27.6. The cells were labeled with 35 S-Promix for 45 min as described above. Thereafter, they were washed twice with phosphatebuffered saline and incubated in 1 ml of DMEM containing 10 mM Met/3 mM Cys for the specified time period. After the chase incubation, cells and media were processed as described above.
Immunoprecipitation of ApoB-Immunoprecipitations were used to quantify the labeled apoB in the cell or secreted into the medium. For this purpose, 100 l of 5ϫ IP buffer was added to each tube containing the conditioned media. Media and cell lysates were incubated at 4°C with gentle rotation for 12 h and then were centrifuged at 10,000 ϫ g for 5 min to remove any unsolubilized proteins or cell debris. The apoB proteins in the resultant media and cell lysates were incubated with rabbit anti-mouse apoB antisera for 6-h at 4°C, and the immunoreaction complex was precipitated with protein A-agarose (Invitrogen, Grand Island, NY). After washing five times with 0.5ϫ IP buffer, the immunoprecipitates were dissolved in SDS-PAGE gel loading buffer and resolved on a 3-12% gradient gel under reducing conditions. The gels were dried, and the radioactivity associated with apoB was quantified on a GS-525 phosphorimaging system using a low beta-screen (Bio-Rad).
Density Distribution of the Newly Secreted ApoB-containing Lipoproteins by Hepatocytes-To determine the density distribution of the apoBcontaining lipoproteins secreted by the liver, hepatocytes isolated from apoB-38.9 or apoB-27.6 heterozygotes and from their wild type littermates were continuously labeled with 35 S-Met/Cys for 3 h in 100-mm dishes (4 ml each) as described above but in the presence of 0.5 mM oleic acids (OA) complexed with bovine serum albumin (ratio of OA to bovine serum albumin ϭ 3.6:1) (35). The media were then subjected to densitygradient ultracentrifugation in a d ϭ 1.006 -1.25 g/ml KBr density gradient (36,37). Fractions were aspirated and dialyzed, thereafter, they were subjected to immunoprecipitation and 35 S-labeled apoBs in each fraction were separated by SDS-PAGE as described above.
Determinations of in Vivo Secretion Rates-Hepatic secretion rates of VLDL triglycerides were measured in littermates of Apob ϩ/ϩ , Apob ϩ/38.9 , and Apob ϩ/27.6 mice (male, 13 weeks old) after intravenous injection of Triton WR-1339 as described (28). Mice were fed a fat-free high carbohydrate diet for 12 h prior to the experiments, and Triton WR-1339 in 100 l of saline (500 mg/kg of body weight) was injected into the mice under light aesthesis with Metophane. Tail vein blood samples were taken at the specified times after injection for triglyceride measurement.
Quantification of Liver Lipid Contents-Lipids were extracted from liver tissues as described previously (38). The dried lipid extracts were dissolved in 1% Triton X-100 in chloroform and dried under a stream of N 2 , and the dried lipid-Triton X-100 complexes were solubilized in H 2 O as described (39) for determination of triglycerides, total cholesterol, and phospholipids using enzymatic kits (WAKO Chemicals, Inc., Richmond, VA). The hepatic lipid concentrations were expressed as micrograms of lipid/mg of protein.
Miscellaneous Procedures-Cellular protein contents were determined using a modified Lowry method (40). Student's t test and analysis of variance were performed to determine the levels of significance of differences.

RESULTS
ApoB-27.6-producing Mice-In our previous study to generate apoB-38.9 mice (28), the homologous recombinant ES cells were stably transfected with a Cre cDNA to express Cre to remove the floxed Neo r cassette from the targeted apoB-38.9specifying allele (28). This step involved a prolonged culture period for the targeted ES cells, and it appeared to result in a low success rate of germ-line transmission. Thus, we wanted to test whether this step is necessary for our targeting construct in which the Neo r cassette was placed in the middle of an intron (i.e. intron 24) (Fig. 1A) and presumably, its retention in the genome may not affect the mRNA expression of the targeted allele. The parental Apob-targeted ES cells (in which the Neo r cassette was not excised) were used directly for blastocystmicroinjection without being subjected to transfection with the Cre expression vector. Two high percentage male chimeras were produced, and both of them were capable of germlinetransmitting the targeted allele. Unexpectedly, however, the resultant engineered mice produced an apoB species with a size close to the previously reported apoB-27.6 (ϳ150 kDa) of FHBL humans (41), instead of apoB-38.9 (Fig. 1B). Nonetheless, Southern blot analysis confirmed that the Apob of these mice were correctly targeted (Fig. 1C).
Characterization of the Targeted Apob Allele mRNA-The The preparation of the targeting construct has been described previously (28). It contains a single nucleotide deletion that leads to production of apoB-38.9. A novel HpaI restriction site was introduced to the mutation site. The predicted structure of the targeted Apob allele is also illustrated (bottom panel). It contained a Loxp-PKG-Neo-Loxp cassette in intron 24. The external probe for Southern blot analysis located immediately downstream of the 3Ј-end of the targeted segment. B, detection of apoB-27.6 by Western analysis. Two microliters of mouse plasma were separated on a 3-12% SDS-PAGE gel. Western blotting was carried out using rabbit anti-mouse apoB polyclonal antibodies as described under "Experimental Procedures." C, a typical Southern blot showing hybridization of 32 P-labeled external probes to HpaI digested-genomic DNA from Apob ϩ/ϩ , Apob ϩ/27.6 , Apob 27.6/27.6 , Apob ϩ/38.9 , and Apob 38.9/38.9 mice. apoB-27.6 in FHBL humans occurred as a result of a donor splice mutation of intron 24 (41), leading to formation of a premature-stop codon 29 residues downstream of the last codon in exon 24 of human Apob (residue 1254) (42). Based on the similarity between the sizes of our mouse-truncated apoB variant and the human apoB-27.6, we suspected that a premature stop codon might have formed near the 3Ј-end of exon 24. To test this possibility, RT-PCR analysis was carried out on liver RNA isolated from two mice homozygous for the targeted allele. The upstream PCR primers corresponded to the 5Ј-end of mouse Apob exon 22 or exon 23, which are external to the targeted construct, whereas the downstream primers tested were chosen from the following region of the targeted Apob allele: 3Ј-and 5Ј-ends of intron 24 of mouse Apob, 5Ј-end of Neo r coding region and 5Ј-end of exon 25 ( Fig. 2A). Of the RT-PCR primer pairs tested, only the exon 22 or exon 23 primer combined with the Neo r primer gave rise to specific RT-PCR products from the Apob 27.6/27.6 liver RNA (920 and 750 bp, respectively) (Fig. 2B). Nucleotide sequencing analysis of these two PCR products revealed that exon 24 of mouse Apob and the coding region of Neo r were spliced together (Fig. 2B). This aberrant apoB RNA splicing lead to formation of a prematurestop codon 6 residues downstream of the last codon of exon 24 and thus producing a truncated apoB-27.6 containing a novel 6-amino acid carboxyl-terminal tail (Fig. 2C). Although the formation of this premature stop codon was an unexpected finding in our current study, it is highly unlikely that one or more other unexpected modifications had also occurred in the coding region of this targeted Apob allele, because the apparent molecular weight of its protein product apoB-27.6 was consistent with the predicted molecular weight of the protein encoded by the full-length apoB-27.6 transcript (ϳ150 kDa). Moreover, removal of the Neo r cassette from this allele gave rise to the expected truncated apoB variant, namely apoB-38.9 (Ref. 28 and Fig. 1B).
Northern analysis of liver RNA using a cDNA probe located near the 5Ј-end of mouse apoB mRNA (encoding aa 26 -289) revealed that there was only one apoB transcript (7.5 kb) in the apoB-27.6 homozygotes (Fig. 3). This transcript, but not wild type apoB-or the apoB-38.9 transcripts, also hybridized to the Neo r cDNA probe (not shown). Together, these data demonstrated the identity of the 7.5-b transcript as the apoB-27.6 transcript. Quantification of hybridization signal revealed that the apoB-27.6 transcript was 3-fold more abundant than the wild type Apob allele transcript (Fig. 3). And, it was 5-fold more abundant than the apoB-38.9 transcript in livers of Apob 27.6/38.9 mice (Fig. 3). Thus, retention of the floxed Neo r in intron 24 interferes with normal apoB RNA splicing and the Cre-mediated excision of Neo r is an indispensable step for our Apob-targeting construct. Nevertheless, this inadvertently generated apoB-27.6-bearing mouse offered us an excellent opportunity to understand the functionality of this small variant of truncated apoB.
Inability of ApoB-27.6 to Support Normal Embryonic Development-The apoB-27.6-heterozygous offspring developed normally and were fertile. However, intercrosses of Apob 27 (Table I), amounting to only 1.8% of all of the offspring, which is 6-fold lower than the percentage (12%, On FPLC analysis, cholesterol peaks corresponding to VLDL (fractions 5-15), LDL (fractions 16 -26), and HDL (fractions 27-42) moderately decreased in the plasmas of apob ϩ/27.6 , compared with wild type mouse plasma (Fig. 4A). All of these lipoprotein cholesterol peaks dramatically diminished in plasma of apob 27.6/38.9 mice. Western blot analysis of the FPLC fractions showed that apoB-27.6 was eluted with HDL-sized particles (fractions 30 -38) (Fig. 4D) that are much smaller than the apoB-38.9 or apoB-48 particles (Fig. 4, B and C).
Hypersecretion of ApoB-27.6 -To compare the secretion rates of apoB-27.6, apoB-38.9, and apoB-48 by the mouse liver, cultured primary hepatocytes isolated from Apob ϩ/ϩ , Apob 38.9/ϩ , or Apob 27.6/ϩ mice were labeled with 35 S-Met/Cys for 3 h, and 35 S-label radioactivity in intracellular and secreted apoB variants was quantified (Fig. 6A). As reportedly previously (28), equal molar amounts of apoB-48 and apoB-38.9 were secreted by the Apob 38.9/ϩ hepatocytes despite less 35 S-labeled apoB-38.9 than 35 S-labeled apoB-48 was accumulated inside the cell (Fig. 6A). In contrast, the relative intensity of the intracellular apoB-27.6 band of Apob 27.6/ϩ hepatocytes was 1.5-fold stronger than that of the apoB-48 (Fig. 6A). More importantly, protein band corresponding to apoB-27.6 in the medium was 5-fold more intensive than that of apoB-48 (Fig.  6A). These data indicated that, on the molar bases, the Apob 27.6/ϩ hepatocytes secreted as much as 7-fold more apoB-27.6 than apoB-48 as the content of Met and Cys residues in apoB-27.6 is only about 70% of that of apoB-48 (based on predictions from the human apoB-100 amino acid sequence). The amounts of apoB-48 secreted by the Apob 38.9/ϩ or Apob 27.6/ϩ cells were ϳ50% of those of the Apob ϩ/ϩ cells.
Pulse-chase studies of Apob 27.6/ϩ hepatocytes revealed that apoB-27.6 was secreted at faster rates and much more effi- a These data were obtained from five litters. b NA, not applicable.  ciently that apoB-48 and apoB-100 from the same cells (Fig.  6B). Thus, the high synthetic rate and the high secretion efficiency of apoB-27.6 both may contribute to the hypersecretion of this truncated variant by the Apob 27.6/ϩ hepatocytes.
To compare the lipid-transporting capacities of apoB-27.6, apoB-38.9, and apoB-48 in our mice, the hepatocytes were labeled with [ 35 S]methionine/cysteine for 4 h in the presence of OA (0.5 mM), and the conditioned media were subjected to density-gradient ultracentrifugation. As shown in Fig. 7, ϳ50% of apoB-48 were secreted as VLDL by hepatocytes of all three Apob genotypes. There were small amounts of apoB-38.9 secreted as VLDL by the Apob 38.9/ϩ hepatocytes, but the majority of apoB-38.9 was secreted as lipoproteins with LDL/HDL densities. In contrast, no apoB-27.6 was detected in VLDL or LDL in the conditioned medium of Apob 27.6/ϩ hepatocytes. Almost all of the apoB-27.6 secreted was distributed at the upper limit of HDL densities, i.e. Ͼ1.15 g/ml. Again, near equal amounts of apoB-48 and apoB-38.9 were secreted by the Apob 38.9/ϩ hepatocytes whereas 6-to 7-fold more apoB-27.6 than apoB-48 was secreted by the Apob 27.6/ϩ hepatocytes. Only small amounts of 35 S-apoB-100 were seen, and they all floated with VLDL densities (Fig. 7).
Impaired Hepatic Triglyceride Secretion and Fatty Livers in the ApoB-27.6-bearing Mice-The in vivo triglyceride secretion rates by livers of Apob 38.9/ϩ , Apob 27.6/ϩ , and Apob ϩ/ϩ mice were determined using Triton-WR1339 as inhibitor for VLDL lipolysis. ApoB-38.9 mutation caused a 35% reduction in hepatic triglyceride secretion rates in Apob 38.9/ϩ mice, whereas the apoB-27.6 mutation produced a 45% decrease in Apob 27.6/ϩ mice (Fig. 8A). Thus, high secretion rates of apoB-27.6 did not offset the inability of apoB-27.6 to transport triglycerides. Due to the lower VLDL-triglyceride secretion rates by the Apob 27.6/ϩ mouse liver, the Apob 27.6/ϩ mice accumulated more triglycerides in their livers than the Apob 38.9/ϩ mice (Fig. 8B). A much more severe fatty liver occurred in the Apob 27.6/38.9 and Apob 27.6/27.6 mice (data not shown).
ApoE Is Not Involved in Rapid Catabolism of Lp-B-27.6 -The high production rates of apoB-27.6 by the hepatocytes and the low levels of apoB-27.6 in the plasma combined strongly suggested that the apoB-27.6-containing lipoproteins were cleared from the plasma very rapidly. To determine if apoE plays a role in catabolism of these lipoproteins, Apob 27.6/ϩ mice were crossbred with apoE knockout mice to disrupt the apoE gene (Apoe). Viable Apoe Ϫ/Ϫ /Apob 27.6/ϩ mice were produced. Disruption of Apoe increased plasma apoB-48 levels in Apob ϩ/ϩ , Apob 27.6/ϩ , and Apob 38.9/ϩ mice by 10-to 15-fold (Fig. 9). It also resulted in a 5-fold increase in plasma apoB-38.9 levels in Apob 38.9/ϩ . In contrast, the absence of Apoe function caused no change in plasma levels of apoB-27.6 in the apoB-27.6 heterozygous mice (Fig. 9), indicating that catabolism of Lp-B-27.6 is apoE-independent.

DISCUSSION
The ApoB-27.6-bearing FHBL Mouse Model-The apoB-27.6 mouse described herein contains a premature-stop codon 6 amino acid residues downstream from residue 1254 of apoB- ApoB proteins in the cell lysate or the medium were immunoprecipitated and resolved on SDS-PAGE (3-12% gel) and quantified using phosphorimaging. The intensities of signals were expressed as arbitrary units (above the gel). B, pulse-chase studies of Apob 27.6/ϩ hepatocytes. Hepatocytes were prepared and labeled for 45 min as described in panel A. Thereafter they were chased for the indicated time periods. After immunoprecipitation and separation on SDS-PAGE, the apoB bands were quantified by phosphorimaging. The lower panel showed a typical gel image that was overexposed to visualize apoB-100. 100. Compared with the previously reported apoB-27.6 in FHBL subjects that contain a novel peptide of 29 residues at the carboxyl terminus (41,42), the apoB-27.6 produced by our mice more faithfully represents the amino-terminal 27.6% of apoB-100. Furthermore, of much value for our studies, the abundance of apoB-27.6 mRNA was considerably higher than that of the wild type allele in the livers of our Apob 27.6/ϩ mice, and of the previously reported apoB-gene-targeted mice (25)(26)(27)(28). Such an elevated level of apoB-27.6 mRNA may be due to the presence of the Neo-resistant gene mRNA in the 3Ј-untranslated region of the apoB-27.6 transcript and thus it may not reflect the apoB-27.6 mRNA levels in FHBL humans. Nev-ertheless, the high hepatic apoB-27.6 mRNA levels lead to augmented synthetic and secretion rates of apoB-27.6 by the hepatocytes of apoB-27.6-bearing mice. Consistent with our results, high levels of hepatic apoB mRNA, such as those occurring in the Apob transgenic mice (43), have been shown to be capable of inducing apoB secretion in vivo, although physiological regulation of apoB secretion occurs mainly at the posttranscriptional levels (44). The availability of our "high apoB-27.6-producer" mice enabled us to study the function and metabolism of apoB-27.6 in vivo.
Inability of ApoB-27.6 to Support Embryogenesis-ApoB is required in the transport of lipids in the yolk sac; in the absence of apoB-containing lipoproteins embryonic development of the central nervous system in mice is grossly deformed, resulting in fetal losses (1,2). The rates of fetal production of Apob 27.6/27.6 mice resembles those of apoB Ϫ/Ϫ null mice (1), suggesting that apoB-27.6 is nearly as deficient in the transport of placental nutrient lipids and in supporting embryonic development as is the absence of apoB. The specific nutrient deficiencies responsible for the fetal wastage remain to be determined. By contrast, yields of Apob 38.9/38.9 offspring are nearly half of expected and yields of apoB 27.6/38.9 offspring are similar to those of apoB 38.9/38.9 , suggesting that the peptide segment between carboxyl termini of apoB-27.6 and apoB-38.9 is critical for embryonic development.
Reduced Ability of ApoB-27.6 to Export Triglycerides in the Liver-The apoB-27.6-apoB-38.9 peptide segment is also important in the export of triglycerides from the liver. In comparison with Apob ϩ/ϩ mice, Apob 38.9/ϩ mice secrete triglycerides at a lower rate into plasma in vivo and accumulate more triglycerides in their livers. Apob 27.6/ϩ mice secrete at an even slower rate and accumulate more triglycerides, despite the high secretion rates of apoB-27.6 protein. These in vivo results are compatible with the notion derived from cell culture experiments that the ability of a carboxyl-terminally truncated form of apoB to transport triglycerides is positively correlated with its size (30,31). In addition, a previous study (45) on transfected hepatoma cells has suggested that the apoB-29-apoB-34 peptide segment may have a critical role in mediating apoB-48-VLDL assembly. Our current study tends to provide in vivo evidence supporting this observation. However, more studies are required to define further the domain(s) within the apoB-27.6-apoB-38.9 segment that may have a particularly important role in determining the lipid-transporting capability of the apoB molecule.
Clearance of ApoB-27.6-containing Lipoproteins and the Role of ApoE-Although the synthesis and secretion rates of apoB-27.6 in our Apob 27.6/ϩ mice were several times higher than those of apoB-100 and apoB-48, apoB-27.6 was present in the plasma at 8-fold lower concentrations than apoB-48 or apoB-100. Similarly, even though the mRNA levels and hepatocellular contents of apoB-27.6 were much higher than those of apoB-38.9, plasma levels of apoB-38.9 were 5-fold higher than those of apoB-27.6 in the Apob 27.6/38.9 mice. Together, these findings strongly suggest that apoB-27.6-containing particles were cleared from the plasma at much faster rates than the apoB-100-, apoB-48-, or apoB-38.9-containing lipoproteins. Moreover, our finding that disruption of Apoe did not cause accumulation of apoB-27.6 in plasma demonstrates that one or more catabolic pathways of apoB-27.6 particles are fundamentally differently from that of apoB-48 particles in which apoE plays a crucial role (23). Unlike apoB-27.6, plasma levels of apoB-38.9 were augmented in response to disruption of Apoe, albeit to a much lesser extent than the apoB-48. Thus, deleting the carboxyl-terminal 490 amino acids of apoB-38.9 may severely impair the ability of the resultant truncated apoB-containing lipoproteins to recruit apoE but still greatly accelerates clearance of these lipoproteins from plasma. Variants of truncated apoB smaller than apoB-31 are not detectable in FHBL humans (12)(13)(14)(15). The results of our present study provide strong evidence that accelerated clearance rates may be responsible for the absence of small truncated apoB variants in FHBL humans, probably via an apoE-independent mechanism.
Due to the low plasma levels of apoB-27.6-containing lipoproteins and the inviability of apoB-27.6 homozygous mice, we have not been able to isolate these lipoproteins from our mice to determine their fractional catabolic rates directly nor study the tissue loci of their catabolism or their cellular catabolic pathway(s). Using other truncated apoB-containing lipoproteins isolated from FHBL humans, we have previously shown that these lipoproteins are cleared from the plasma faster than the normal apoB-100-containing LDL (22,24). Renal uptake via the proximal tubular endocytic receptor megalin was identified as a major mechanism underlying the accelerated catabolism of these lipoproteins (22). We have localized a megalin-binding site within the amino-terminal 15% of human apoB-100 (46). Thus, it is entirely possible that the renal megalin-mediated pathway is also an important pathway for catabolism of the apoB-27.6-containing particles. The molecular size and/or the shape of a plasma protein are important determinants in its renal glomerular permeability. The circulating mouse apoB-27.6 lipoproteins were much smaller than apoB-100-, apoB-48-, or apoB-38.9-containing lipoproteins. This may explain why apoB-27.6 and, presumably, other small variants of truncated apoB were metabolized faster than the latter. However, there may be one or more other mechanisms involved in uptake of the lipoproteins containing small variants of truncated apoB. For example, a study by Kreuzer et al. (47) indicated that a peptide FIG. 9. Western blot analysis of apoB in apoB-38.9 and apoB-27.6 mice in apoE wild type or apoE-null background. Plasma was sampled from 8-week-old mice after fasting for 4 h. A Western blot was performed as described under "Experimental Procedures." Each lane represents plasma samples pooled from three mice of each genotype. The experiment was repeated three times, and similar results were obtained. segment within the amino-terminal 23% of apoB-100 could be recognized by a macrophage-scavenger receptor without oxidative modification of the protein. This scavenger receptor-recognition site appeared to be inactive in the native apoB-48-or apoB-100-containing lipoproteins (47) but could be active in the unmodified small truncated apoB-containing lipoproteins. Further studies are required to elucidate the relative importance of particle sizes, apoE recognition, or other factors in determining the differing rates of clearance of apoB-27.6 and apoB-38.9 lipoprotein particles.