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THEMES
Children's Foundation Research Center of Memphis, Le Bonheur Children's Medical Center, Department of Pediatrics, University of Tennessee Health Science Center, Memphis, Tennessee
Submitted 30 April 2007 ; accepted in final form 7 May 2007
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ABSTRACT |
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 TOP ABSTRACT Key Proteins in Chylomicron...Chylomicron AssemblyChylomicron Assembly in the...Chylomicron Trafficking in...Role of HNF-4 in...Summary and Directions for...GRANTSREFERENCES |
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apolipoprotein A-IV; apolipoprotein B-48; microsomal triglyceride transfer protein; newborn; small intestine
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Once the products of lipid digestion enter the enterocyte, they must traverse the cytoplasm to the endoplasmic reticulum (ER), where the resynthesis of complex lipids occurs. Fatty acid binding proteins (FABPs) in the enterocyte exist in two forms: liver FABP (L-FABP) and intestinal FABP (I-FABP). In particular, L-FABP may function to translocate long-chain fatty acids and MAG from the apical membrane to the ER. Experimental evidence from intestinal isografts implanted subcutaneously into adult mice suggests that spatial and temporal induction along the small intestine is genetically programmed and not triggered by dietary lipid, although dietary lipid may modulate the degree of expression (17). Fatty acids and MAG are utilized to resynthesize first to diacylglycerol and then TG by acyl-CoA:monoacylglycerol acyltransferase (MGAT) and acyl-CoA:diacylglycerol acyltransferase (DGAT), respectively, in the ER. This monoacylglycerol pathway predominates in the small intestine, including that of the neonate. A recent advance has been the cloning and characterization of mouse and human MGAT and DGAT isoforms and their regulation (4). Previous studies, mainly in the developing rat, have suggested that enterocyte fatty acid esterification is not limiting in the newborn and suckling period (19). Furthermore, dietary lipid appears to be the most important modulator for the development and maintenance of intestinal fatty acid esterification.
The focus of this review will be the recent elucidation of the developmental aspects of the intracellular processes associated with chylomicron assembly in the ER and regulation of the key proteins involved in this complex process.
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Key Proteins in Chylomicron Assembly |
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TOPABSTRACTKey Proteins in Chylomicron...Chylomicron AssemblyChylomicron Assembly in the...Chylomicron Trafficking in...Role of HNF-4 in...Summary and Directions for...GRANTSREFERENCES |
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Apo B-48.
Apo B exists as two distinct forms. Apo B-100 is the larger protein, produced primarily by the pre- and postnatal liver and prenatal small intestine, and contains the low-density lipoprotein receptor-binding domain. The smaller form, apo B-48, is primarily synthesized by the postnatal small intestine and lacks the low-density lipoprotein receptor-binding domain. Both are produced from the same gene by a unique posttranscriptional mRNA editing complex, apobec-1, that creates a premature stop codon by deamidation of a single base (C 
U) in the apo B-100 mRNA transcript that allows coding for apo B-48 that correlates with the amino-terminal half of apo B-100. Apo B-100 is essential for very-low-density lipoprotein (VLDL) assembly in hepatocytes, and apo B-48 is requisite for chylomicron assembly in enterocytes.
MTP.
MTP, a heterodimeric protein complex possessing lipid transfer activity, functions in the small intestine and liver to transport ER membrane-bound lipid, primarily newly synthesized TG, to newly translated apo B in the ER lumen as the first step in TG-rich lipoprotein biogenesis (Fig. 1). This initial lipidation of apo B prevents proteosome-mediated degradation. In the small intestine, MTP may also facilitate the further lipidation of nascent chylomicrons beyond the first apo B rescue step. MTP is a heterodimer consisting of a large subunit (97 kDa), which possesses the lipid transfer activity, and a smaller subunit identical to protein disulfide isomerase (PDI; 55 kDa). Mutations in the MTP large subunit are responsible for the disease abetalipoproteinemia, which is characterized by an inability to secrete intestinal chylomicrons or hepatic VLDL. As a component of MTP, PDI appears to maintain the large subunit in soluble form and may play a role in targeting MTP to the ER lumen. Since PDI is ubiquitous and abundant in the ER, it is not thought to be a limiting factor in MTP function.
Apo A-IV.
Apo A-IV is a lipid binding protein that is expressed predominantly in the mammalian small intestine. Although numerous extraintestinal functions have been ascribed to apo A-IV (21), the preponderance of evidence points to a primary role in intestinal lipid absorption. Of all the intestinal genes associated with lipid absorption, the apo A-IV gene is the most responsive to intestinal lipid flux. In the enterocyte, apo A-IV is incorporated into nascent chylomicrons at an early stage of biogenesis in the ER and is secreted on the surface of chylomicrons at the basolateral membrane. Thereafter, most apo A-IV dissociates from the chylomicron surface and, in humans, circulates predominantly as a lipid-free protein.
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Chylomicron Assembly |
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TOPABSTRACTKey Proteins in Chylomicron...Chylomicron AssemblyChylomicron Assembly in the...Chylomicron Trafficking in...Role of HNF-4 in...Summary and Directions for...GRANTSREFERENCES |
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The nascent chylomicron exits the ER in a specialized vesicle, the prechylomicron transport vesicle (PCTV), for transport to the cis-Golgi. Data from the adult rat have suggested that budding of the PCTV is the rate-limiting step in lipid absorption (20). The ER to Golgi trafficking of chylomicrons appears to be unique from that of other cargo, such as proteins. Recent data from Neeli et al. (16) have indicated that L-FABP can select cargo for and bud the PCTV from the ER membrane. The budding is independent of coating protein II (COPII) proteins, but trafficking and fusion with the cis-Golgi are dependent on COPII proteins, Sar1 and Sec23/24, as well as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) fusion complex composed of vesicle-associated membrane protein 7 (VAMP7), syntaxin 5, Bet1, and vti1a. In the Golgi, apo B-48 glycosylation is modified, lipid composition is altered, and the particle acquires apo A-I. Subsequently, the particles undergo exocytosis at the basolateral membrane.
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Chylomicron Assembly in the Newborn Intestine |
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TOPABSTRACTKey Proteins in Chylomicron...Chylomicron AssemblyChylomicron Assembly in the...Chylomicron Trafficking in...Role of HNF-4 in...Summary and Directions for...GRANTSREFERENCES |
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Developmental regulation of apo B-48.
Apo B-48 in the developing small intestine is regulated in a manner that optimizes chylomicron formation. Apobec-1 expression is upregulated before parturition in the small intestine in rodents, swine, and humans to switch from apo B-100 production in the fetal gut to apo B-48 in the neonatal intestine (23). Interestingly, in apobec-1 knockout mice, in which the gut produces only apo B-100, there is a subtle defect in chylomicron secretion that may be of particular importance in the face of high lipid flux in the neonate, thus making the timing of the switch important (24).
In the adult rat, the mesenteric lymphatic output of apo B-48 is relatively constant regardless of the amount of absorbed lipid. Given that there is one apo B molecule per chylomicron, it appears that the adult rat secretes the same number of chylomicrons regardless of lipid load but can increase chylomicron size with increasing fat absorption. In contrast, we have observed in both the suckling swine (2) and in cultured newborn pig intestinal epithelial (IPEC-1) cells (8) that with fatty acid absorption, especially unsaturated 18-carbon fatty acids, jejunal apo B-48 mass increases, likely due to decreased targeted posttranslational degradation rather than increased synthesis, leading to increased apo B-48 secretion. This increase would make more apo B-48 available for a greater number of particles to enhance lipid export capacity. In animal studies, this effect was observed in both the jejunum and ileum, suggesting the participation of the distal small intestine in dietary lipid absorption in the newborn, perhaps to accommodate absorption of the large lipid load that may escape proximal absorption. In vivo, the magnitude of this induction diminishes near weaning. Biliary diversion strikingly downregulated apo B mass below baseline nonlipid-fed levels in both the jejunum and ileum, underscoring the importance of biliary secretion in the maintenance of apo B-48 production in the newborn. This may have clinical relevance in infants with cholestatic liver diseases that lead to decreased bile flow.
Developmental regulation of MTP.
To date, there has been a paucity of information on the developmental expression of MTP in the small intestine. MTP large subunit expression has been analyzed in the fetal mouse using in situ hybridization (18). These studies suggest an important role for MTP in mobilization of yolk sac lipid early in gestation prior to liver and gut expression. Also, MTP expression has been studied in the early gestation human small intestine (12). In these studies, MTP large subunit mRNA, protein, and activity were detected in a decreasing proximal to distal gradient, but enzymatic activity was low in vitro and unresponsive to increasing substrate lipid.
To define the developmental pattern of expression of MTP large subunit mRNA and protein in the developing swine model, samples of the small intestine and liver were collected from 40-day-old gestation fetal, 2-day-old newborn, 3-wk-old weanling, and 2-mo-old weaned female swine (13). In fetal animals, MTP mRNA expression was high in the intestine and liver. However, an unexpected finding was a relatively high level of intestinal MTP large subunit mRNA expression in the fetus in the face of low levels of protein of the appropriate apparent molecular weight. The dissociation of mRNA and protein levels suggests that MTP large subunit protein levels may be regulated at the translational level in the fetal pig intestine.
Postnatally, jejunal MTP large subunit expression paralleled the intake of a high-fat breast milk diet and declined after weaning. Ileal expression was comparable with that of the jejunum in 2-day-old animals but declined to low levels afterward. Postnatal levels of MTP large subunit protein generally paralleled mRNA expression. The 24-h intraduodenal infusion of a high-TG diet in 2-day-old animals acutely increased jejunal and ileal MTP large subunit mRNA levels compared with a low-TG diet. The induction of ileal expression may have been due to some degree of ileal lipid absorption in newborn animals receiving a high-fat diet, which may have exceeded the absorptive capacity of the proximal small intestine. Thus, early increased ileal MTP large subunit expression may represent an important compensatory mechanism to scavenge proximally unabsorbed lipid.
We have studied the roles of glucocorticoids and fatty acids in MTP regulation in a newborn swine enterocyte cell line (IPEC-1) (13). Dexamethasone did not play a major role in the regulation of MTP expression in differentiating IPEC-1 cells. In contrast, fatty acids either upregulated or downregulated MTP expression depending on the specific fatty acid and duration of exposure.
These results suggest that expression of the MTP large subunit gene in the developing swine small intestine follows a pattern suited to accommodate the lipid absorptive requirements of the animal. Although programmed genetic cues undoubtedly play a major role in the developmental regulation of MTP gene expression, clearly the amount and fatty acid composition of dietary lipid also play important regulatory roles.
Developmental regulation of apo A-IV.
We have previously shown that apo A-IV is expressed in the small intestine of neonatal swine (3). In animals given a high-fat intraduodenal lipid infusion for 24 h, jejunal apo A-IV mRNA expression increased sevenfold compared with control animals given a low-fat infusion (3). This observation, coupled with the fact that this lipid responsiveness decreased as the piglets were weaned from a high-fat breast milk diet, suggested that apo A-IV plays a role in facilitating intestinal lipid absorption in the suckling newborn. Weinberg et al. (22) have proposed that the presence of relatively hydrophilic helices in apo A-IV, compared with other apolipoproteins, enables apo A-IV to adopt an elastic, pressure-sensitive conformation at lipid interfaces. During chylomicron assembly, these hydrophilic 
-helices may be recruited into the expanding nascent particle surface and, by maintaining constant interfacial elasticity, would stabilize particle growth and enable the packaging of more core lipid molecules per particle.
To test the hypothesis that expression of apo A-IV enhances enterocyte chylomicron secretion in response to fatty acid absorption, we developed a stably transfected newborn swine intestinal epithelial cell line that synthesizes and secretes excess swine apo A-IV. We chose the IPEC-1 cell line for this purpose specifically because these cells have relatively low lipid transport efficiency and low background apo A-IV expression compared with native enterocytes, enabling us to observe marked increases in lipid export should they occur. Using this cell line, we demonstrated that when cultured with oleic acid (OA; C18:1n-9), high-level constitutive overexpression and secretion of apo A-IV enhanced radiolabeled basolateral TG and cholesteryl ester secretion by fivefold and phospholipid by twofold, mainly as lipoproteins in the chylomicron and VLDL density range (15).
A limitation of this approach was the fact that the level of constitutive apo A-IV overexpression was far higher than the normal physiological range. To address this issue, IPEC-1 cells were transfected with a tetracycline-regulatable transactivator and full-length swine apo A-IV cDNA driven by the tetracycline response element, allowing regulation of apo A-IV expression and basolateral secretion in a physiological range by culture medium doxycycline concentration (14). To identify protein structural components essential for this enhancement, we also performed similar experiments with human apo A-IV as well as truncated mutants of human apo A-IV resembling swine and chicken apo A-IV.
In cells incubated with OA, a dose-response relationship was observed between medium doxycycline concentration and basolateral apo A-IV and TG secretion. Similarly, regulated expression of another apolipoprotein produced in the small intestine, apo A-I, did not enhance lipid secretion. The mean diameter of TG-rich lipoproteins secreted from doxycycline-treated cells was larger than from untreated cells (87.0 vs. 53.4 nm). Using the same expression system, full-length human apo A-IV (376 amino acids); a "pig-like" human apo A-IV, lacking the carboxy-terminal EQQQ repeats found in human apo A-IV (361 amino acids); and a "chicken-like" apo A-IV, further truncated to 343 amino acids, were expressed in IPEC-1 cells. With increasing protein secretion, cells expressing the full-length human apo A-IV displayed a 2-fold increase in TG secretion; in sharp contrast, cells expressing the pig-like human apo A-IV displayed a 25-fold increase in TG secretion and a corresponding increase in lipoprotein diameter. When human apo A-IV was further truncated to yield a chicken-like protein, TG secretion was inhibited.
These experiments demonstrated that swine apo A-IV enhances basolateral TG secretion in a dose-dependent manner by increasing the size of secreted lipoproteins. These data suggest that the region in the human apo A-IV protein from residues 344 to 354 is critical to its ability to enhance lipid secretion, perhaps by enabling the packaging of additional core TG into chylomicron particles. Since this region is not a lipid binding domain, this enhancement may be due to interaction of this domain with other regions of apo A-IV or other proteins that may influence the tertiary structure of apo A-IV and the lipid binding affinity of its -helical domains. The EQQQ-rich carboxy terminus of human apo A-IV may play an inhibitory or modulatory role in chylomicron packaging in humans. The truncated human apo A-IV is probably more efficient in promoting chylomicron secretion than swine apo A-IV due to its more hydrophilic -helices.
These studies established that physiological levels of native swine and human apo A-IV secretion enhance TG transport by inducing the secretion of larger chylomicron/VLDL particles, either by biophysically stabilizing their surface or possibly by interacting with apo B to slow their passage through the cellular compartments where second-step expansion occurs (7). Taken together, these data suggest that although, unlike apo B, apo A-IV is not obligatory for intestinal lipoprotein assembly, it may facilitate maximally efficient lipoprotein secretion. This function may be especially important in the newborn, where the intestine must rapidly and efficiently absorb the large lipid load provided by breast milk. Human apo A-IV mutant studies have established the importance of the carboxy-terminal sequences of apo A-IV in the enhancement of chylomicron secretion, probably by influencing particle assembly and/or lipidation in the ER. The exact biophysical properties of apo A-IV and the intracellular mechanisms by which it mediates this effect require further study.
These data may appear to conflict with the observations from transgenic mouse models indicating that lipid absorption was unaffected either by overexpression of human apo A-IV or targeted disruption of the apo A-IV gene. However, these animals were not evaluated under conditions of sustained lipid absorption that approached the rate-limiting capacity of the enterocyte. Moreover, inactivation of apo A-IV synthesis in transgenic animals may induce alternate compensatory pathways that maintain normal levels of intestinal lipid absorption. In this regard, the suckling rat does not efficiently transport dietary lipid in lymph chylomicrons, and indirect evidence has suggested that the portal venous route may be important in this species and possibly in all rodents, including mice, during the suckling period (5). In contrast, lymphatic transport is important for lipid absorption in the suckling swine (1), a species whose intestinal development more closely resembles that of the human.
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Chylomicron Trafficking in Newborn Enterocytes |
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TOPABSTRACTKey Proteins in Chylomicron...Chylomicron AssemblyChylomicron Assembly in the...Chylomicron Trafficking in...Role of HNF-4 in...Summary and Directions for...GRANTSREFERENCES |
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Some insight into the trafficking process in the immature enterocyte has been gained from in vitro studies of trafficking of labeled lipid from ER to Golgi vesicles performed in IPEC-1 cells (25). These studies demonstrated that trafficking efficiency varies with different fatty acids with stearic acid (C18:0) and the long-chain polyunsaturated fatty acid (LC-PUFA) eicosapentaenoic acid (EPA; C20:5n-3), adversely influencing ER to Golgi TG trafficking compared with OA. Furthermore, EPA inhibited ER lipid synthesis and transfer of TG from the ER membrane to ER luminal particles. These results suggest that the fatty acid composition of infant formula should be balanced between nutrient needs and enterocyte absorptive efficiency. In particular, these results may have relevance to the recent addition of the LC-PUFAs to infant formula for their positive effects on brain and eye development.
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Role of HNF-4 in Neonatal Lipid Absorption |
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TOPABSTRACTKey Proteins in Chylomicron...Chylomicron AssemblyChylomicron Assembly in the...Chylomicron Trafficking in...Role of HNF-4 in...Summary and Directions for...GRANTSREFERENCES |
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. This transcription factor regulates numerous genes involved in glucose and lipid metabolism, including apo A-IV and MTP, and is capable of ligand-dependent and -independent transactivation, although the identity of physiological ligands is controversial. We recently demonstrated coordinate upregulation of both HNF-4 as well as apo A-IV and MTP expression by acute lipid absorption in the newborn swine jejunum and by fatty acid treatment in IPEC-1 cells (11). Fatty acid-independent overexpression of HNF-4 in IPEC-1 cells induced apo A-IV and MTP expression at the transcriptional level, and fatty acid treatment did not further enhance this transcriptional activation. These data suggest that the acute induction of the apo A-IV and MTP genes by dietary lipid in the newborn intestine may occur, at least in part, via ligand-independent transactivation by HNF-4, which is itself induced by a lipid-mediated mechanism. Thus, HNF-4 may serve as a "master switch" for coordinating chylomicron assembly in the newborn. |
Summary and Directions for Future Research |
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TOPABSTRACTKey Proteins in Chylomicron...Chylomicron AssemblyChylomicron Assembly in the...Chylomicron Trafficking in...Role of HNF-4 in...Summary and Directions for...GRANTSREFERENCES |
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Rather than static descriptive studies of gene expression during development, more studies of the active physiological regulation by dietary factors in neonatal lipid absorption are needed. In particular, studies of the developmental and dietary regulation of PCTV budding and ER to Golgi trafficking are needed. This is especially true if the budding process is also the rate-limiting step in neonatal lipid absorption. The neonatal swine model, which provides "industrial-strength" quantities of cells and tissue, and whose gut development is close to that of the human infant, is an ideal model for such studies.
As we learn more of the regulation of these processes in the immature intestine, especially at the transcriptional level, agents that induce expression of apo B-48, MTP, and apo A-IV as well as key molecules involved in PCTV budding and trafficking would have potential therapeutic value in increasing the lipid absorptive efficiency of the small intestine in premature infants or those with diseases such as short bowel syndrome. Also, greater knowledge of the fatty acid specificity of these processes would guide the optimal lipid composition of infant formula. Finally, determining whether these processes are susceptible to metabolic imprinting early in life may have implications for later diseases such as hyperlipidemia and obesity.
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GRANTS |
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TOPABSTRACTKey Proteins in Chylomicron...Chylomicron AssemblyChylomicron Assembly in the...Chylomicron Trafficking in...Role of HNF-4 in...Summary and Directions for...GRANTSREFERENCES |
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FOOTNOTES |
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TOPABSTRACTKey Proteins in Chylomicron...Chylomicron AssemblyChylomicron Assembly in the...Chylomicron Trafficking in...Role of HNF-4 in...Summary and Directions for...GRANTSREFERENCES |
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