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Original Article |
Correspondence to: Robert L. Hamilton.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMaterials and MethodsRESULTSDISCUSSIONREFERENCES |
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The mechanisms for packaging large quantities of neutral lipids into apolipoprotein (apo) B-containing lipoproteins (chylomicrons or VLDL) are incompletely understood. However, several lines of evidence have suggested that the addition of core lipids to apoB to form a lipoprotein particle within the endoplasmic reticulum (ER) may involve two steps: first, the addition of small amounts of core lipids to membrane-bound apoB, generating a lipid-poor, small apoB-containing particle, and second, the fusion of that particle with a larger, independently formed triglyceride-rich and apoB-free "lipid particle." We sought to test this two-step hypothesis of apoB core lipidation by using electron microscopy to compare chylomicron assembly in mice that are genetically deficient in the ability to synthesize apoB in the intestine to control mice. In 19-day gestational mice (fasting setting) that were deficient in intestinal apoB synthesis, chylomicron-sized lipid particles in the lumen of the enterocyte ER were even more abundant and were 2- to 3-fold larger than those in the enterocytes of normal control mice. However, there were fewer lipid-staining particles in the Golgi apparatus, and many fewer particles in the extracellular space, compared with normal control mice. In both types of newborn suckling mice, much larger lipid particles were assembled within the lumen of the ER. They were however, less abundant and rarely reached the Golgi apparatus in fatty enterocytes of intestines deficient in apoB synthesis.
These observations provide in vivo evidence that chylomicron formation could involve the synthesis of apoB-free triglyceride-rich particles within the endoplasmic reticulum (ER) lumen, and that the transport of these lipid particles out of the ER to Golgi apparatus and interstitium is facilitated by the acquisition of apoB.—Hamilton, R. L., J. S. Wong, C. M. Cham, L. B. Nielsen, and S. G. Young. Chylomicron-sized lipid particles are formed in the setting of apolipoprotein B deficiency. J. Lipid Res. 1998. 39: 1543–1557.
Supplementary key words: absorptive enterocytes, lipoproteins, intestinal fat absorption, endoplasmic reticulum, Golgi apparatus, cytosolic lipid droplets
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMaterials and MethodsRESULTSDISCUSSIONREFERENCES |
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Gage and Fish (1) established the concept that dietary fats, and not proteins or carbohydrates, caused the appearance of "a multitude of glancing particles which look like motes in a sunbeam" when intestinal lymph or peripheral blood was viewed with the aid of a "dark-field ultramicroscope." They termed these lipid-containing particles "chylomicrons," from the Greek chylos for chyle and mikron for any minute object. Since these seminal studies of fat absorption were published nearly 75 years ago, we have learned that the core of chylomicron particles contains large amounts of triglycerides and that the surface protein components of chylomicrons (now called apolipoproteins) play critical roles in the assembly, secretion, and metabolism of these particles (2).
Apolipoprotein B (apoB) is known to have a particularly critical role in the assembly of the triglyceride-rich lipoproteins. ApoB-48 is used for chylomicron assembly in intestinal enterocytes (2) (3) (4). ApoB-100 is used for VLDL assembly in visceral yolk sac endoderm (5); and either apoB-100 or apoB-48 is used for VLDL formation in hepatocytes (2) (3) (4). We have been interested in understanding the assembly of the triglyceride-rich lipoproteins, particularly the mechanisms by which the triglyceride core is added to apoB during the assembly of these lipoproteins.
Previously, we described the absence of apoB on lipid particles of VLDL size within the smooth endoplasmic reticulum (SER) of rat hepatocytes assessed by immunoelectron microscopy (6). These findings, taken together with subsequent kinetic studies of VLDL assembly and secretion (7) (8) (9) (10) (11) have led to the hypothesis that the production of nascent VLDL involves the synthesis of a triglyceride-rich (and apoB-free) lipid particle in the SER, which joins a lipid-poor apoB-particle formed in the rough ER (RER) before the completed lipoprotein particle is secreted from the cell. We call this the "two-step hypothesis" for apoB core lipidation (12) (13) ( Figure 1). In the current study, we sought to further assess this two-step hypothesis, and in particular to determine whether the second-step triglyceride (TG)-rich particles can form in the ER lumen independently of apoB. To approach this issue experimentally, we have used electron microscopy to characterize chylomicron assembly in mice that are deficient in intestinal apoB synthesis.
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Our laboratories have generated mice deficient in intestinal apoB synthesis by mating human apoB transgenic mice (in which the transgene was expressed only in the yolk sac and liver, but not in the intestine) with apoB knockout mice (14) (15). The human apoB transgenic mice that were homozygous for the knockout mutation (HuBTg+/oApob-/-) develop normally in utero, and at birth are normal in size, weight, and physical appearance. However, because these mice made no apoB (mouse or human) in the intestine, they could not synthesize chylomicrons and developed fat malabsorption and vitamin E deficiency. Within 24 h of birth, these mice developed a white, protuberant abdomen caused by a massive accumulation of intracellular fat within the intestinal epithelium, from the duodenum to the colon (15). Within 2 days of birth, marked growth retardation was evident. Because of their small size, we have dubbed these mice `runts.'
Our ultrastructural analysis of the absorptive enterocytes of the 2-week-old runts revealed that the villus enterocytes were essentially "filled" with cytosolic lipid droplets; the cells had little ER, and no membrane-bound triglyceride-rich particles were observed (15). Ultrastructural studies by others have revealed that the duodenal enterocytes of the villus tips of 10 day suckling rats are fatty and largely lacking in chylomicrons, whereas mid-villus enterocytes are not fatty and contain nascent chylomicrons within the secretory organelles (16). Based on these studies, we hypothesized that an ultrastructural analysis of the runts' intestines might yield valuable insights into lipoprotein assembly if the analyses could be performed prior to the massive accumulation of cytosolic fat droplets.
In this study, we examined the ultrastructure of runt duodenal enterocytes at 19 days of gestation (i.e. in the fasting setting) when apoB expression and lipoprotein secretion are established, but before the mice have taken their first fatty meal. We also examined the duodenum of runts during the first few hours after birth (within hours of their first fatty meal). The principal goal of these studies was to determine whether TG-rich particles are formed in the enterocyte ER lumen in the setting of apoB deficiency.
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Materials and Methods |
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TOPABSTRACTINTRODUCTIONMaterials and MethodsRESULTSDISCUSSIONREFERENCES |
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ApoB knockout mice
ApoB knockout mice were generated previously by gene-targeting in mouse embryonic stem cells (14). In the absence of the human apoB transgene, mice that were homozygous for the apoB knockout mutation (Apob-/-) died early during embryonic development, likely because the absence of VLDL synthesis in the yolk sac interferes with the delivery of lipid nutrients to the developing embryo (5).
Human apoB transgenic mice and mice deficient in apoB expression in the intestine
To generate mice deficient in intestinal apoB, the apoB knockout mice were mated with human apoB transgenic mice in which the transgene was expressed in the liver and yolk sac, but not in the intestines (17) (18) (19). For these studies, we used human apoB transgenic mice (HuBTg+/o, line 1102) generated with the P1 bacteriophage clone, p158 (18). The p158 human apoB transgene was not expressed in the intestines because it lacks the DNA sequences that control the intestinal expression of the apoB gene (18) (19) (20). To obtain mice deficient in intestinal apoB synthesis, we intercrossed HuBTg+/oApob+/- mice to obtain human apoB transgenic mice that were homozygous for the mouse apoB knockout mutation (HuBTg+/oApob-/- or HuBTg+/+ Apob-/-). The lethal developmental abnormalities observed in Apob-/- mice could be rescued with p158 because that clone confers human apoB expression in the yolk sac during development (14). However, because the HuBTg+/oApob-/- mice could not make apoB (mouse or human) in the intestine, they developed severe fat malabsorption and vitamin E deficiency and grew very slowly (15). Because of their small size, those mice have been dubbed `runts' in our laboratory. For simplicity's sake, we have also referred to them as runts in this paper.
In this paper, we have characterized runts and littermate control mice after 19 days of gestation, and within a few hours after birth. The genotypes of the control mice consisted of HuBTg+/o Apob+/-, HuBTg+/oApob+/+, and Apob+/- mice. To obtain 19-day gestational mice, timed matings of HuBTg+/oApob+/- mice were performed as previously described (5). The 19-day embryos were genotyped by Southern blot analysis of tail DNA. The DNA probes used to detect the presence of the apoB knockout mutation and to detect the presence of the human apoB transgene have been described previously (14) (18).
Western blots and RNase protection assays
To assess the presence of mouse and human apoB in the plasma of runts and control mice, 2 ml of plasma was size-fractionated on a 4% polyacrylamide-SDS gel; the separated proteins were then transferred to a sheet of nitrocellulose membrane for Western blot analysis with a rabbit antiserum specific for mouse apoB (20) or a monoclonal antibody (1D (1)) (21) (22) specific for human apoB. Antiserum binding was detected with a peroxidase-labeled goat anti-rabbit or goat anti-mouse Ig and the enhanced chemiluminescense detection reagents (Amersham, Arlington Heights, IL).
To detect the presence of human apoB mRNA in mouse tissues, RNA was prepared from both liver and duodenum of p158-transgenic mice. As controls for these experiments, we examined the liver and intestinal RNA from human apoB transgenic mice and 19-day embryos generated with a 145-kb bacterial artificial chromosome clone, BAC(70,22) (19). BAC(70,22) contained 70 kb of 5' flanking sequences and therefore contained all of the sequences controlling appropriate intestinal expression of the human apoB gene (19). RNase protection assays of apoB mRNA levels were performed using a 121-bp fragment of exon 1. The RNase protection experiments were performed with the RPA II ribonuclease protection assay kit (Ambion, Austin, TX) according to the manufacturer's instructions. Electrophoresis of protected RNA fragments was performed on 6% polyacrylamide gels containing 7 mol/L urea (Novex, San Diego, CA). The dried gels were exposed to autoradiographic film.
Preparation of mouse tissues for electron microscopy
Pregnant mice at 19 days of gestation were anesthetized by an intraperitoneal injection of pentobarbital (~2 mg/25 gm body weight). A midline abdominal incision was made, and the uterine wall was opened around individual fetuses. As each pregnancy produced 5–10 pups, fixation and surgical removal of individual fetal mouse duodenal and upper jejunal tissue took several hours. During this time, the mother was kept under a warming lamp and carefully monitored, a gauze sponge drenched in warm saline covering the incision. Each pup was removed from the uterine cavity, its tail removed for genotyping, its abdominal wall incised, and its viscera exposed. Under a dissecting microscope, a 27-gauge needle was inserted into the duodenal lumen, and the small intestine was flooded with a fixative of 1.5% glutaraldehyde, 4% polyvinylpyrolidone, and 0.5% calcium chloride in 0.1 M sodium cacodylate buffer, pH 7.4 (5) (15) from a syringe. Simultaneously, fixative was delivered drop-wise for 10 min onto the serosal surface, submerging the intestine in a pool of fixative in the abdominal cavity.
For early suckling mouse intestines, pups born the previous night were genotyped and their intestines were fixed during the afternoon according to the techniques described above. Thus, these pups had been suckling for approximately 5–12 h.
To stain the core lipids of lipoproteins, we used a modified version of the imidazole-buffered osmium tetroxide procedure described by Angermüller and Fahimi (23). Depending on the anticipated fat content of the tissue, we used 2–4 different concentrations of imidazole, ranging from 0.02 M to 0.3 M. We also added a 2% aqueous uranyl acetate block stain for 1 h at 4°C before dehydration and embedding in Epon (DuPont, Wilmington, DE). Thin sections were stained for 5 min with 0.8% aqueous lead citrate and photographed in a Siemens 101 electron microscope (Siemens CTI Corp., Knoxville, TN).
To be certain that electron microscopic images were representative of a particular mouse genotype, a minimum of three pups of each genotype and at each time point were examined. In addition, 2–6 blocks of tissue from each mouse pup were thin sectioned and photographed. Finally, duodenal and upper jejunal enterocytes were examined throughout the tip, middle, and basal regions of the villi in all cases. Although we present for illustration only selected fields that the authors agreed were representative of each situation, more than 800 electron micrographs were printed and examined for this project.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMaterials and MethodsRESULTSDISCUSSIONREFERENCES |
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Analysis of human and mouse apoB expression in the intestines
`Runts,' which are homozygous for the apoB knockout mutation and carry the p158 human apoB transgene, were identified by Southern blot analysis ( Figure 2). We have shown that mouse apoB is not expressed in embryos that are homozygous for the apoB knockout mutation (14) (15) (19). We could not detect the presence of mouse apoB in the plasma of adult `runts' by Western blot analysis ( Figure 3). We also could not detect mouse apoB-100 in the plasma of adult runts with a monoclonal antibody-based RIA specific for mouse apoB-100 (data not shown). We have also shown that human apoB is essentially undetectable in the intestines of adult human apoB transgenic mice generated with the P1 bacteriophage clone, p158 (19) (24). In this study, we performed RNase protection assays to determine whether human apoB gene expression is also absent in the intestines of 19-day gestational transgenic mice generated with p158. As shown in Figure 4 of the 19-day p158-transgenic embryos, human apoB expression was robust in the liver but was either undetectable or extremely faint in the intestines. Similarly, in adult p158 human apoB transgenic mice, human apoB expression was robust in the liver but was extremely faint or undetectable in the intestine (data not shown). As controls for these experiments, we analyzed human apoB expression in 19-day gestational BAC(70,22) transgenic mice. BAC(70,22) contains the distant 5' intestinal enhancer element and confers full expression of the apoB gene to the intestine (19). In the BAC(70,22) mice, there was robust expression of the human apoB gene in both the liver and intestine (Figure 4).
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Ultrastructure of the Golgi apparatus of duodenal absorptive enterocytes of 19-day gestational mice
We analyzed lipoprotein assembly in the villus enterocytes of `runts' at 19 days of gestation because of prior data indicating that lipoprotein secretion would be developed at that time point.2 We concentrated our analyses on the duodenal villi distal to the entrance of the common bile duct and the upper jejunum. Triglyceride-rich lipoprotein assembly and secretion occurs in the apical portion of absorptive enterocytes between the nucleus and microvillus border. As shown in Figure 5, low magnification electron micrographs revealed a consistent difference between control and runt enterocytes. In the absorptive enterocytes of control mice, we often observed large numbers of nascent chylomicrons within numerous supranuclear Golgi compartments. In contrast, the Golgi compartments of runt absorptive enterocytes often completely lacked lipid-staining particles (Figure 5B). However, Golgi areas infrequently contained several lipid-staining particles (Figure 5B, top), but generally only one or two large particles of chylomicron size were observed. These larger lipid particles in runt enterocytes were most commonly found singly, and were closely adjacent to, but not clearly within the Golgi stack ( Figure 6A and 6B). In contrast, smaller chylomicrons were often clustered together clearly within forming Golgi secretory vesicles in control enterocytes (Figure 6A).
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Ultrastructure of the endoplasmic reticulum (ER) of absorptive enterocytes of 19-day gestational mice
In the apical cytoplasm beneath the microvillus border, lipid-staining particles of chylomicron size were evident within the lumenal spaces of the membranes of the ER of both control and runt duodenal absorptive enterocytes ( Figure 7). However, the ultrastructural characteristics of ER lipid-staining particles were distinctly different in two respects. First, the ER lipid particles from the runts were clearly more abundant and easier to find; and second, the particles were always substantially larger than those observed in controls (Figure 7). The mean diameter of the lipid-staining particles within the lumen of the ER of the runts was more than double the diameter of the ER lipid particles in the `apoB-expressing' control enterocytes (1,772 ± 627 Å in the runts versus 725 ± 235 Å in control mice) ( Table 1).
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Ultrastructural analysis of lipoproteins within the extracellular space of 19-day gestational mice
Numerous lipid-staining particles of nascent chylomicron size were frequently observed between adjacent absorptive enterocytes of control mice ( Figure 8A). In contrast, the lipid-staining particles were much less frequently observed in the extracellular spaces of the runts. However, whenever lipid-staining particles were observed in the extracellular spaces, they were invariably larger than those observed in controls. Figure 8B shows an unusually large number of large-sized extracellular lipid particles in runt intestine. (Although most of the other micrographs revealed few or no extracellular lipoproteins, this image was chosen to demonstrate the larger size of lipid-staining particles in the extracellular spaces of the runt intestines.)
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Thus, the diameters of lipid-staining particles in the ER, Golgi apparatus, and interstitium were uniformly smaller in controls compared to runts (Table 1). As we were only able to identify and measure a few particles in the extracellular spaces of runts' intestines, it is difficult to draw firm conclusions regarding the comparison of particles sizes in the extracellular space.
Ultrastructure of the ER and Golgi apparatus of absorptive enterocytes of newborn suckling mice
The apical cytoplasm of duodenal enterocytes of control newborn suckling mice is characterized by the presence of numerous lipid-staining particles within the lumenal spaces of the ER ( Figure 9A). These TG-rich particles are 4- to 8-times greater in diameter (4,000 Å to 6,000 Å) than those of the control intestine at 19 days of gestation (~750 Å) (contrast Figure 7A and Figure 9A). In the newborn control mice, the duodenal enterocytes at the villus tips sometimes also contained large cytosolic lipid droplets in addition to the lipid particles within the lumenal spaces of the ER and Golgi apparatus (data not shown). However, in newborn runts, the villus tip enterocytes were almost always fatty. Apical cytoplasm was filled with very large (5–10 micron) cytosolic lipid droplets and very few cytosolic organelles remained ( Figure 10A). In particular, the quantity of ER was greatly reduced in the villus tip enterocytes of runts, and it was difficult to find ER that contained lipid particles. Thus, the ultrastructural appearance of the villus tip enterocytes of newborn suckling runts was closely similar to enterocytes that we observed previously in the 2-week-old suckling runts (15). Interestingly, enterocytes along the sides of the villi in the newborn suckling runts (away from the villus tips) exhibited a different ultrastructural appearance. Although cytosolic lipid droplets were present to a variable extent, these runt enterocytes often contained abundant ER membranes with content lipid particles in the lumenal space (Figure 9B and Figure 10B). These TG-rich particles within the lumen of the ER of suckling runt enterocytes were less numerous than in the control enterocytes, but were of comparable size (compare Figure 9A and Figure 9B). Large lipid-staining particles were often observed singly within the membrane bound lumenal spaces of the Golgi apparatus of enterocytes of control newborn suckling mice ( Figure 11A), indicating that they are nascent chylomicrons bound for the extracellular spaces. In contrast, most Golgi compartments of runt suckling absorptive enterocytes lacked these nascent chylomicron particles, although a few single lipid particles were sometimes observed (Figure 11B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMaterials and MethodsRESULTSDISCUSSIONREFERENCES |
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We reported previously that lipid-staining particles of VLDL size within the lumenal spaces of the SER of rat hepatocytes lacked apoB, as judged by immunoelectron microscopy (6). In those studies, the ER apoB was localized to the RER, where no lipid particles of VLDL size were observed. These ultrastructural observations led to the concept that apoB obtains its core lipids in two separate steps during the assembly of TG-rich lipoproteins, as depicted in Figure 1. The basic features of this hypothesis are that apoB-free TG-rich particles are generated within the ER lumenal space, and that these particles then join with a small, lipid-poor apoB-containing particle before exiting the cell. Within the past few years, this concept has been supported by pulse-chase studies of lipoprotein assembly in cultured rat McA-RH7777 hepatoma cells. These studies demonstrated that small apoB-containing HDL particles within the lumen of the ER could be transformed, apparently in a single rapid step, into VLDL particles simply by incubating the cells with oleate (8) (9) (10) (11).
We elected to study runt (HuBTg+/oApob-/-) mice at 19 days of gestation because our prior studies had shown that it was next to impossible to examine the ultrastructure of chylomicron assembly during the late suckling phase, in which the absorptive enterocytes of runts were virtually filled with cytosolic fat droplets, and the quantity of ER within these cells was markedly reduced or even absent.3 At 19 days of gestation, the runts and control mice were of identical size. The maturity of the absorptive enterocytes in runts and controls was evidently equivalent, as judged by the appearance of the microvillus border and by the fact that they apparently contained equivalent amounts of ER and other organelles within the apical portions of the enterocytes. However, with respect to the ultrastructure of chylomicron assembly, the control enterocytes and runt enterocytes were different. First, the number of lipid-staining particles within the lumen of the ER was often greater in the runt enterocytes compared with the controls. Second, the lipid-staining particles were always substantially larger in the runt enterocytes. Third, the number of the larger lipid-staining particles in the Golgi apparatus of runt enterocytes was substantially reduced. Fourth, these larger lipid particles were often adjacent to and apparently not within the Golgi stacks, and fifth, the number of lipid-staining particles outside of runt enterocytes was strikingly reduced. Whereas we often observed numerous smaller-sized lipid particles in extracellular compartments of the control animals, we found only a few instances in which the larger lipid particles were evident outside of enterocytes in the runts.
How should this series of ultrastructural findings be interpreted? The greatly decreased numbers of lipid-staining particles in the extracellular spaces outside of the runt enterocytes compared to controls was the most constant finding. This is certainly consistent with the long-standing concept that apoB synthesis is required for secretion of lipoproteins from cells (2) (3) (4). An extremely sensitive RNase protection assay suggested that trace levels of apoB synthesis may well exist within the absorptive enterocytes of the 19-day runts (as a result of transgene leakiness), and we therefore believe it is possible that the rare extracellular particles that we observed in the runts could actually contain a molecule of apoB. However, the appearance of the ER in runt enterocytes is particularly interesting. In the ER, we found increased numbers of lipid particles, despite markedly decreased numbers of particles in the Golgi apparatus and extracellular spaces. We believe that this observation supports the two-step assembly model of apoB core lipidation for lipoprotein synthesis. Our explanation for these observations is that the formation of lipid particles within the ER does not require apoB synthesis, but that their transport out of the cell is greatly facilitated by apoB synthesis. We propose that the vast majority of the lipid particles within the ER represent the apoB-free "second-step" particles (depicted in Figure 1). These putative second-step particles were also substantially larger in the enterocytes of runts than control mice. We suggest that the much larger size of these particles is due to a retarded rate of transport through the ER compartment as a consequence of apoB deficiency. In other words, we suggest that the lipid particles are assembled in the ER in the absence of apoB, and that an apoB molecule is responsible for allowing them to be swept towards and into the Golgi compartment and out of the cell. When an apoB molecule does not come along (or comes along very rarely), as in the case of the runts, the ER lipid particles continue to accumulate lipids within the ER lumen and become larger.
Our ultrastructural observations show that many fewer of these large lipid particles in runts appear to `leak' through the Golgi apparatus to the extracellular spaces compared to wild-type intestine, which deserves special comment. Adult runts that survived suckling and thrived on low fat chow diet had extremely low plasma levels of vitamin E and mildly reduced levels of essential fatty acids (15). It seems possible that a `leakage' of small numbers of lipid particles throughout the length of the small intestine may be sufficient to ensure some fat absorption, preventing overt manifestations of vitamin E and essential fatty acid deficiency. Interestingly, this phenotype in the runts is quite similar to that in humans with abetalipoproteinemia, which is characterized by trace levels of apoB-containing lipoproteins in the plasma (25) and only a minimal reduction in plasma levels of essential fatty acids (26). We suspect that the `leakage' of a comparatively small number of lipid particles into the bloodstream in abetalipoproteinemia may help to explain the absence of essential fatty acid deficiency in this disorder, and may also explain why these individuals on a low fat diet respond favorably to high doses of orally administered vitamin E (2) (3) (4) (25).
From a teleological standpoint, a two-step process for adding core lipids to chylomicrons makes sense, especially as a diet rich in fats has little or no effect on apoB expression itself. When dietary fats are abundant (e.g., the suckling phase), the lipid particles within the lumen of the ER can become larger, leading to larger chylomicron particles. If the `core' diameter of first-step apoB-containing particles is ~100 Å, the amount of core volume would be increased by 1000-fold by adding a second-step particle that was ~1000 Å in diameter and by 8000-fold if the second-step particle were ~2000 Å (13). Thus, the ability to modulate the size of lipid particles allows an organism to increase the size of apoB-containing TG-rich lipoproteins when nutrients are plentiful and to decrease their size during periods of fasting. We suspect that the ability to add additional lipids to apoB-containing lipoproteins may prove to be unique to the intestine and the liver. For example, we have not observed lipid particles within the ER of yolk sac visceral endoderm cells from Apob-/- mice (5). However, this epithelial cell contains only RER and is devoid of SER.
Whereas the present studies provide in vivo support for the two-step hypothesis of apoB core lipidation, we emphasize that many questions remain regarding the molecular mechanisms for this process, particularly the molecules involved in the formation of the ER lipid particles. For example, hypothesized gene products that would presumably be needed for the generation of apoB-free lipid particles within the lumen of the ER have not yet been identified. Also, the precise role of microsomal triglyceride transfer protein (MTP) in the core lipidation of apoB-containing lipoproteins is not established. It seems likely that MTP is essential for the generation of `first step' particles, inasmuch as virtually no apoB-containing lipoproteins are secreted from cells in the absence of MTP (27). However, the role of MTP in the second step of apoB core lipidation requires further study. In pulse-chase studies of apoB assembly in McA-RH7777 cells, one research group concluded that MTP inhibitor drugs had little effect on the addition of bulk lipids to dense apoB48-containing particles (first-step particles) within the lumen of the ER, implying that MTP might not play a critical role in the second step (10). However, this conclusion has been disputed by another group of investigators who also used pulse-chase studies to study lipoprotein assembly in McA-RH7777 cells (11).
In the future, we predict that ultrastructural studies of yolk sac, liver, or intestinal cells may help to delineate the role of MTP in lipoprotein assembly. We have recently generated and characterized MTP-deficient mice (30) with phenotypes analogous to the apoB knockout mice (5) (14). Homozygotes died early in development, the visceral yolk sac endoderm was fatty and lacked ultrastructural evidence of VLDL assembly/secretion, and the embryos manifested neurodevelopmental abnormalities. We suspect that tissue-specific knockout strategies or transgene rescue strategies (akin to the one used in the current study) will allow us to be able to study the ultrastructural characteristics of lipoprotein assembly in tissues lacking MTP.
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FOOTNOTES |
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2 We chose the 19-day time-point because intestinal villi develop in rats on the 18th day of gestation and lipoprotein particle assembly/secretion is first observed on the 19th day of gestation (28). 
3 The reason for the striking reduction of runt enterocyte ER in the presence of massive amounts of cytosolic fat is not known. During the first hours of suckling, the villus tip cells had a massive accumulation of cytosolic fat droplets, much reduced amounts of ER, and no ER lipid particles. The mid-villus enterocytes of runts were often not as fatty and had abundant lipid particles within the ER lumenal space, suggesting that the cellular machinery for generating lipoproteins may be down-regulated by excessive cytosolic fat. Interestingly, the extent of Golgi membranes did not appear to be significantly reduced in runt absorptive enterocytes containing massive amounts of cytosolic fat droplets.
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ACKNOWLEDGMENTS |
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Supported by National Institutes of Health grants HL41633, HL14237PO1, HL14237, and a Grant-in-Aid from the American Heart Association, California Affiliate. L.B.N. was a recipient of a postdoctoral fellowship award from the American Heart Association, California Affiliate.
Manuscript received 12 February 1998.
Abbreviations: apoB, apolipoprotein B; ER, endoplasmic reticulum; SER, smooth ER; RER, rough ER; MTP, microsomal triglyceride transfer protein; TG, triglycerides; VLDL, very low density lipoproteins; HDL, high density lipoproteins
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMaterials and MethodsRESULTSDISCUSSIONREFERENCES |
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L. Washington, G. A. Cook, and C. M. Mansbach II Inhibition of carnitine palmitoyltransferase in the rat small intestine reduces export of triacylglycerol into the lymph J. Lipid Res., July 1, 2003; 44(7): 1395 - 1403. [Abstract] [Full Text] [PDF]
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J. A. Sellers, L. Hou, H. Athar, M. M. Hussain, and G. S. Shelness A Drosophila Microsomal Triglyceride Transfer Protein Homolog Promotes the Assembly and Secretion of Human Apolipoprotein B: IMPLICATIONS FOR HUMAN AND INSECT LIPID TRANSPORT AND METABOLISM J. Biol. Chem., May 23, 2003; 278(22): 20367 - 20373. [Abstract] [Full Text] [PDF]
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