The 1991 Borden Award Lectureel Selected aspects of intraluminal and intracellular phases of intestinal fat absorption EMILELEVY"

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/11/14 For personal use only.

Research Centre, HdpH'taQP1 Ste-Justine Q& DepaPbmeut of Nutrition, Universitd CB& Montrdak, MontrdaH (Qugbee), C a d Received November 29, 1991 LEVY,E. 1992. The 1991 Borden Award Lecture. Selected aspects of intralufinal and intracellular phases of intestinal fat absorption. Can. I. Physiol. Phamcol. 70: 4 13 -4 19. The recognition of chylomicrons as dietary lipid transporters dates back to more than '90 years and marks a milestone in lipoprotein history. Conventionally, three phases constitute the process of absorption of exogenous fat: intrduminal, intestinal, and delivery. The intrdumiml phase includes chemical hydrolysis by lipolytic enzymes and the micellar solubilHation of lipolytic products by bile acids. The intestinal phase comprises the difision of miceUes through the unstirred water layer, passive diffusion across the microvillous membrane of the enterocyte, sand the fornation of lipid-carrying lipoproteins. The delivery phase involves the exocytosis of chylomicrons from the absorptive cells and their subsequent removal by lymphatic structures md the systemic circulation. The precise steps and factors involved in all phases of chylornicron synthesis are not yet known, but both experimental and clinical studies have been helpful. Of the inborn metabolic disorders, the prerequisite function of apoligoprotein (apo B) for the assembly and release of lipoprotein particles stood out. Moreover, evidence emerged that the enterscyte produces apo B-1W in addition to apo B-48. Calcium and essential fatty acid status originates as determinants for trdglyceride-rich particle synthesis. Furthermore, the developmental changes and regulatory factors of lipoprotein elaboration represent excellent tools in the study of the intracellular mechanisms of lipid transport. Key words: intestinal fat absorption, chylomicron, lipoproteins and apoproteins, ontogeny, essential fatty acid deficiency, calcium. LEVY,E. 1992. The 1991 Borden Award Lecture. Selected aspects of intrdumiml and intracellular phases of intestinal fat absorption. Can. J. Physiol. Phamcol. 70 : 413 -419. La reconnaissance des chylomicrons c o m e transporteurs de lipides remonte ii plus de '90 ans et constihe rane Chpe dCterminante &ins l'histoire des lipproteines. Conventionnellement, on rkpartit Be processus d'sabsorption des graisses exogknes en trois phases: les phases intraluminale, intestinale et de sCcr6toire. La phase intrduminale inclut une hydrolyse chimique par les emzymes liplytiques aimi que la solubilisation micellaire des produits Bipolytiques par les acides biliaires. La phase intestinde est caractkriske par la diffusion des micelles ii travers la couche aqueuse won remuke, la diffusion passive ii travers la membrane des microvillositks de I'entCrocyte et la formation de lipoprotc5ines transporteuses de lipides. La phase de &crktoire implique l'exocytose des chylomicrgbns des celldes absorbantes et leur incopration subskquente par les structures Bymphatiques et la circulation systkmique. On ne connait pas encore toutes les 6tapes et les facteurs pdcidment impIiqu& dans les phases de la synthbse des chylomicrons, les Ctudes expkrimentales et cliniques y ayant toutefois sapport6 quelques Cclaircissements. En ce qui conceme les erreurs congCnitdes du mktabolisme, ces ktudes ont fait ressortir que la fonction de l'apo B h i t prkrquise pour le regroupement et la libkration des particules de lipoprotkines. La production par l'entdrocyte de I'apo B-100 en plus de cdle de H'apo B-48 a aussi CtC mise en Cvidence. Il ressort aussi que 1'Ctat du calcium et des acides gras essentiels sont des facteurs d6teminstnts pour la synthbse des particules riches en triglyckrides. De plus, les variations dCveloppementaleset les facteurs rkgulateurs de la composition des 1ipprotCines constituent d'exce~lentsoutils pour 19Cude des m6canismes intrstcellulaires du transport lipidique. Mots el& : absorption de graisses intestinales, chylornkrons, 1ipoprotCines et apoprotkines, ontoghie, carence en acides gras essentiels, cdciurn. [Traduit par la rCdsaction]

Atherosclerosis with its potentidly f a d complicatiorms have motivated numerous scientific workers to channel their energies in the field of lipid research. In recent years, considerable efforts have k e n directed towards the undersinding of Bipoprotein metabolism. The liver has been a primary focus of attention, in view of its role as the major site of the manufacture of endogenous lipids. Various experimental hepatic models, including liver perhsion and hepatocyte culture, have greatly facilitated studies on the hepatic delivery and regulation of lipoprotein metabolism. However, the gut dso plays a pivotal role in lipoprotein 'The Borden Award Lecture of the Canadian Society for Nutritionid Sciences was delivered at the annual meeting of the Canadian Federation of Biologicd Sciences, Kingston, Ont., June 1 1, 1991 , and has undergone the Journal's usual peer review. 2Address for correspondence: Pediatric Research Centre, HBpital Ste-Justine. 3175 C6te Sae-Catherine. MontrCal (Qukkc), Canada ., H3T 1C5. ,

Printed in Canada I Imprim&au Canada

mtabolism, Nevertheless, despite intensive research on the overdl scheme of digestion and absorption of dietary fat, littae is known about the factors that regulate the intracellular assembly, transport, and secretion of lipoproteins by enterocytes. The lack of an adequate intestinal model has hampered advancement in &is important area of research. The p u v s of this report is to highlight selected aspects of intestind lipid absorption, focusing on new findings and illustrating their place in the increasing3y complex field of intestinal lipid processing.

Lipids and intraluminal digestion Lipids available for absorption are derived from both dietary and endogenous origins. Triglycerides (TGs), the major lipids account for 40% of the energy source of our typical Western diet. Endogenous phospholipids and cholesterol, derived from b i l i a ~and other sources such as sdiva and desquamated cells, dso constitute intrdumind lipids available for uptake and processing. Irrespective of the 9

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/11/14 For personal use only.

414

CAN. J. PHYSIOL. PHARMACOL. VOL. 30, 1992

particular physicochemicd properties of lipids, hydrophobic or mpkipathic, they form a group of compounds that requires very complex processes for their digestion and absorption. TriglyceHides consist of a molecule of glycerol esterified with three fatty acid moieties. The fatty acid composition is predominantly high in saturates and monounsaturates. In their native form, TGs cannot be transported into the cell, owing to heir size and hydrophobic characteristics. In order for a triglyceride molecule to be absorbed, it must first undergo hydrolysis md then solubilization of the hydrolytic products (Levy et al. 1988; Thomson and Dietschy 1981). Digestion of TGs begins in the stomach by two enzymes, gastric and lingual lipase (Hamosh 1979; Levy et d. 1981; Levy et d. 1982). These emymes are active in the prepyloric region at an acid pH, and continue the lipolytic process in the smdl intestine. They hydrolyze medium-chain TGs present in significant concentrations (8- 10%)in human milk more rapidly than longchain TGs. Medium chain fatty acids are dso partly absorbed by the stomach directly (Levy et d. 1984). Thus, the stomach plays an important role in the digestion of fat, especially in the context of physiologicd or pathological pancreatic insufficiency (neonates or cystic fibrosis patients) (Levy et d.1991d; Roy et d. 1988). However, it should be pointed out that most of the digestion of TGs, cholesterol esters, and phospholipids occurs in the upper smdl intestine, under the concerted action of biliary and pancreatic secretions. Hydrolysis of long-chain TGs is achieved by pancreatic lipase, which requires the pancreatic cofactor, colipase, for its stabilization and optimal hydrolytic activity (Gsrstrorn m d Erlanson 2 97 1). Other important hydrolytic enzymes that significantly contribute to catalyze the detachment of fatty acid from phospholipids and esterified cholesterol are phospholipase A2 and cholesterol esterase, respectively. The interaction of appropriate amounts of bile acids results in micellar dispersion of lipolytic products, assuring their shuttle from the bulk water phase across the unstirred water layer to the region adjacent to the cell surface (Thomson and Dietschy 198h ; Westergmrd and Dietschy 1976).

Mneosal uptake and intracdlular process Many of the products of lipid digestion (2-monoglyceride, lysophosphatidylcholine, free cholesterol) detach from bile sdt rnicelles because of the low pH microcompartment (Daniel et d. 1985; Shiau et al. 19851, and cross the unstirred water layer and brush border membrane by passive absorption. Their translocation from the brush border membrane to the endoplasmic reticulum is mediated by a fatty-acid-binding protein (FMP) (Ocher et al. 1972). The concentration of this protein is greater in the villus tip enterocytes than in the crypts, and is greater in the jejunum compared with the ileum (Ocher et al. 1972; Ocher et d. 1974w, 19748). HABP also has a greater affinity for unsaturated fatty acids (Ocher et al. 1972; Ocher et al. 1974b). Besides directing fatty acids to the endoplasmic reticulum, it also has the important hnction of enhancing the activation of fatty acids (acyl CoA synthesis), which catalyzes the first step in the intraeellular resynthesis of TG (Glatz md Veerlamp 1985). After interiorization into the smooth endoplasmic reticulum, fatty acids and monoglycerides are reesterified to TGs (Johnston 1978; Thomson and Dietschy 1981). Similarly, free cholesterol is reassembled to estet-iified cholesterol (Field 1984; Haung and Nomm 1976) and lysophosphatidylcbobe to phosphatidyl~holine~These

reactions are catalyzed by specific enzymes located at the cytoplasmic surface of the smooth endoplasmic reticulum membrane (Field 1984; Haung and Nomm 1976). These lipids then migrate through the eisternae sf the smooth endoplasmic reticulum to the rough endoplasrnie reticulum, and with the addition of approkins, prechylomicron particles are formed (Tso and GoHlamudi 1984). These nascent chylomicrons are then transported to the Golgi apparatus for their final assembly. Glycosylation initiated in the rough endoplasmic reticulum is completed in the Golgi apparatus (Green and Glichan 1981). Finally, extrusion of the completed chylo~cronsinto the intercellular space occurs subsequent to the hsion of Golgi vesicles containing well-individualized chylomicrons with the lateral plasma membrane (Sabesin 1976; Sabesin and Frase 1977). Intestinal very low density lipoproteins (VLBL), which are formed mostly in the fasting state to carry endogenous lipids (Widmueller and Levy 19681, probably follow the same biosynthetie pathway. In addition to chylornicrons and VLDL, the third class of lipoproteins synthesized by the enterwyte is the high density lipoprotein (HDL), which is the smallest and the densest lipoprotein (Green et d. 1978).

Apolipoprotein ( a p B) is a large hydrophobic glycoprotein found in plasma in heterogeneous sizes (Kme 1983). Apo B-100, a 549-kDa protein, is an essential component of VEDL and LBL (Goldstein and Bmwn 1977; Kane 1983). Apo B-48, a smaller protein of 264 kDa, is a major surface constituent of chylomicrons (Herbert et d. 1983; Kane f 983). Both types of a p B are amphipathic proteins required for the assembly and secretion of TG-rich lipoproteins. Indeed, inherited disorders of apo B deficiency, owing either to a simple structural modification or to a defect in their secretion, result in abnormalities in lipoprotein elaboration, and consequently , fat mdabsorption ( k v y et al. 8987a, 1987b). In humans, the higher molecular weight apo B-100 was believed to be synthesized exclusively by the liver, and aps B 4 8 only by the intestine (Herbert et d. 1983). In our laboratory, we have combined high resolution imunoelectron microscopy with polyclond and monoclonal antibodies specific far aps B-1043 and aps B-48 to determine heir iaatracellular localization (Levy et al. 1998b). Our data revealed that, at the electron microscopic level, both aplipoproteins B- 100 and B 4 8 are present in subcellular compartments of the human enterocytes, including apical smooth membrane vesicles, the Golgi region, and basslaterd membrane, suggesting the synthesis of both forms of apo B by intestinal epithelial cells. To test this hypothesis, human intestine was incubated with [3H]Teucine, homogenized, a d subjected to immunoprecipitation for apo B (Levy et d. 1990b)-When examined by polyacrylamide gel electrophoresis, two peaks were found at the positions of B- 100 and B48. The predominant form appeared to be apo B48. Taken together, our data suggest that the human intestine is able to synthesize and express apo B-100 in addition to apo B 4 8 (Levy et al. 1998b). This novel finding has great potential significance in terms of atherogenesis. A p B- lMpbut not a p B-48, is recognized by receptors in the vascular endotheliurn, dlowing LDL apo B-1W to contribute to atheroma formation (Goldstein and B m m 1977). Our current research is now focusing on dietary measures that modulate the expression of apo B-100 versus apo B-48 by the enterocyte.

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/11/14 For personal use only.

LEVY

Recently, the presence of an in-frame stop codon in intestin d mRNA for apo B-100 has been reported. Chen et al. (1987), Powell d d. (1987), a d Hospatmger et d e(1987) demonstrated that apo B 4 8 is producd fmm the aps B-1W gene by a novel mechanism involving mRNA editing. In exmining the intestinal a p B cDNA, they discovered that a single 8: -. U base change is introduced at h e nucleotide position 6666 sf apo B-100 mRNA, resulting in a change of the ne (CAA). This nucleotide substitution yields an in-frame stop codon (TAA/UAA), which results in apo B-48 production. Our findings relative to the presence of apo B-100 in adult human intestine indicate that a modulation of apo B mRNA may take place. The factors affecting this regulation remain to be established. However, it should be pointed out that the aps B mRNA editing has recently been shown to be modulated by fasting and refeeding, as well as by thyroid hormone in the rat liver, h o w n to produce both apo B48 and a p B- 100 (Davidson et d. 1990). Additiond studies are d s o necessary to exmine the ontogeny of apo B d N A editing given that recent studies have reported h a t the synthesis of a p B-100 in fetal intestine progressively decreased during maturation (Glichan et idbB. 1986). The ultrastmcturd alocdization of the two forms of aplipoprotein B was achieved in our lab by applying the protein A -gold immunocytmhernicd techniques (Levy et d * 1990b) in combination with highly specific monoclond antibodies. These studies reveded labelling over the microvilli, a p i d smooth membrane vesicles, and the trans Golgi region. The labelling on the apical region of the cell could be assigned to an internalization phenomenon of apo B-100 by the epitheli d cells. After internalization, aps B-100 could be transported within smooth membrane vesicles or multivesicular bodies and then transported to the &lgi cistemae. The intense labelling in the apical area of the enterwyte, reflecting internalization activity, does not fit the classical theory of chylomicron formation (Cardell et d. 1967; Redgrave 1983; Tytgat et d, 19'71). Microvilli, apical smooth membrane vesicles , multivesicular bodies, as well as the trans Golgi cistemae are subcellular compartments traditionally involved in endscytotic activity (Pastan m d Willingham 1985). Whether the internalized exogenous a p B is incorporated into the chylomicron pathway remains to be determined.

Fatty acid deficiency m d intestinal lipprotein synthesis Until recently, essentid fatty acid (EFA) deficiency in humans was regarded as an extreme rarity. Using recent, more sensitive techniques, the existence of EFA deficiency has now been demonstrated in several high risk groups, including elderly patients with peripheral vascular disease, prolonged fat-free intravenous feeding, patients with severe trauma and bums, low fat combined with high protein diets, and hyperlipidemia (Holman 1968). Recendy, we have developed an accurate m d sensitive method for the measurement of fatty acids in plasma using the triene/tetraene ratio (Lepage et d. 1989). Using this standard as a definition of EFA deficiency, we documented a high incidence of EFA deficiency in patients with cystic fibrosis ( h p a g e et d. 1989). In a subsequent study, we demonstrated that the abnormalities in lipoprotein composition, size, and metabolism in cystic fibrosis patients are, in part, related to their EFA deficiency (Levy et al. 1989b). Since cystic fibrosis EFA deficiency is generally accom-

415

panied by other metabolic defects, there is no clear understanding of the role sf EFA status on the composition and metabolism of lipprsteins. Thus, we have chosen to use an animal model of EFA deficiency to improve our insight into the mechanisms involved (Levy et d.1990~2).Our results have shown increased concentrations of free fatty acids, TGs, total cholesterol, free cholesterol, and phospholipids in EFA deficient rats compared with pair-fed controls (Levy et d. 1990a). Moreover, post-heparin extrahepatic lipoprotein lipase activity was significantly decreased, a d could account for the hypertriglyceridernia as well as for the relative triglyceride enrichment of VLDL, intermediate density lipprotein, and LDL particles. This enzymatic depletion of lipoprotein lipase was mainly due to the adipose tissue compartment, since a higher level of hepatic lipase was found in acetone extracts of liver. N&ough essential fatty acid deficiency causes pathological changes in the liver, kidney, lung, and testes (Holman 1968), little is known concerning its effect on the digestive system. However, decreased weight gain has been noted in EFAdeficient young mirnds (Holman 19668), and a large study of infants fed skim milk based diets concluded that EEFA deficiency resulted in loose stools (Hansen et d q1963). The elucidation of the processes that limit growth in EFA deficiency is lacking. We have performed studies to evaluate the effect of EFA deficiency on intrdurnind factors involved in the digestive phase m d on the intracellular events responsible for the synthesis and delivery of lipoproteins (Levy et d.1992a). Our results reveal h a t EFA deficiency impaired both the extracellular and intracellular mechanisms responsible for n o m d lipid absorption (Levy et d.1992a). When plasma TG concentrations were memured at hourly intervals after an o r d fat load in rats, the percent increase of TG over fasting values was significantly lower in EFA-deficient rats compared with controls. Similarly, a reduced plasma chylomicron concentration was noted in EFA-deficient rats (Levy et d. 1992a). After documenting impaired absorption of lipids, we focused on the concomjiQmtevents governing the lipid absovtive pathway in EFA deficiency. We first examined i n t r d u i n d factors, including lipase, bile composition, and bile acid secretion (Levy et d. 1992a). Pancreatic hgase activity was not affected by EFA deficiency. However, after canndating the common bile duct and collecting the bile from animals with and without EFA deficiency, a significant decrease was recorded in bile flow, bile acid secretory rate, bile cholesterol, and bile phospholipids. Equally important are the intracdlular events leading to the formation and secretion of chylomicrons. To determine whether these processes are affected by EFA deficiency, we employed jejunal organ culture m e & d s (Levy et d o1992~~). Lipid and lipoprotein synthesis by explants were studied by the addition of ["Cjoleic acid. A reduction sf the synthesis md secretion of both intestinal chylorr5cron a d VLDL was noted in EFA deficiency. What t t e the mechanisms respnsible far the different abnormalities observed in EFA-deficient animals? HDL is the cholesterol-carrying lipoprotein in rats. It is d s s the greferentid source of cholesterol for the synthesis of bile acids (Schwartz et d.19'78). In other studies, we reported that EEFA deficiency results in a trend toward lower concentrations of HDL fractions (Levy et d. 2989; Levy et d. 1990a). This HDL reduction codd result in a reduced transfer of cholesterol from HDE to the liver for bile salt synthesis. Further-

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/11/14 For personal use only.

416

CAN. J. PHYSIBL. PHARMACBL. VOL. '90,1992

more, a p E isoforms are closely associated with HDL particles and play an intricate role in their metabolism (Mhley et al. 1984). Along with others (Ney et al. B987), we have noted that a p E~ electrophoretic mobility is altered in EFAdeficient rats (Levy et d. 1890a). These alterations me in concert with a decrease in cholesterol transfer for bile acid synthesis. More difficult to explain are the effects of EFA deficiency on chylomicron synthesis. Nevertheless, it has k e n shown that the degree of fatty acid saturation influences the mucosd lipid cornpsition (Yurkowski et al. 1970), affects intestinal plasm membrane fluidity ((Brasitus et al. %985),and consequently results in functional alterations of membrane emymes such as microvillus phosphatase or basolateral Nag/Kf ATPase (Brasitus et d. 1985). It is likely that EEFA deficiency alters the dynamic structure of the intestinal mucosa that continually undergoes biochemical, morphological, and ultrastructural changes (hblond 1981). The short life span of the renewing villous cells makes the cell membranes of the intestin d mucosa with their high csncentrations of linoleic (1189 n -6 ) and arachidonic acid (20 :4 pt -6) (Christen et a1. 1989) particularly sensitive to changes in dietary lipids (Hjelte et d. a 990).

Cdcinm and intestinal lipoproteins Intestinal fat absorption is a complex process influenced by a number of intrdtminal m d intracellular factors. One such factor h a t we have recently studied is the role of cdcium on fat absoqtion and on intracellular processes including lipid synthesis and secretion. The experimental protocol was particularly difficult t s elaborate, since calcium is ubiquitous, as a necessary element for intracelldar and extracellular physiologic processes (Ramussen 1986). Even for in vitro studies including cell or organ culture, cdciurn is essential to maintain viability. Given that cdcium enters the cell through specific cdcium chmnels, we took advantage of a new potent c d c i m channel blocker to determine the effects of calicium depletion on intestinal fat absorption (Levy et al. 1992). Three experimental strategies were elaborated to investigate the effect of clentimem, a calcium c h n e l blocker, on intestinal fat absorption: fat meal test, mesenteric lymph cannulation, and intestinal organ culture. We initidly determined the fat absorption of rats treated with clentiazem (Levy et d. 1992). A fat meal was delivered into the duodenum by tube feeding, and blood was drawn from the jugular vein before the fat loading md post-prandidly for TG determinations. A similar fat med test was performed for control rats that had not been pretreated with clentiazem. Our results demonstrated that post-prandid triglyeeridemia was lower in the clentiazem-treated group, with significantly decreased TG concentration at 2 and 3 h. Furthermore, a nearly identical pattern of decreased percentage output was d s o found for chylomicrons, indicating reduced intestinal fat absorption in clentimem-treated rats. Since plasma TG concentrations are determined by a variety of non-intestinal factors, including hepatic secretion, hydrolysis by lipoprotein lipase, and uptake by receptor processes, mesenteric lymphatic cannulation was performed to specificdly determke the intestind TG delivery and thereby define the effect of clentimem on lipid absorption (Levy et d. 1992). The volumes of lymph flow and TG output were measured prior to and post intrdumind administration of clentiazem in

a group of rats h a t received a fat meal intraduodenalal%y.No significant differences were observed in lymph flow. Qn the other hand, a marked f d in TG secretion was noted in treated rats. Similarly, a reduction in chylomicron and TG output was measured in the lymph of treated rats. To determine whether clentiazem has a direct effect on TG biosynthesis, we studied these processes in vitro using cultured jejunal explants (Levy et d. 11992). The incorporation of f"V&leic acid into TG was diminished by the addition of the dmg to the medium. The reduction of TG synthesis mounted to about 28% of the basd vdues with low concentrations of clentimem (10- 100 pg/mL), whereas 807% inhibition was noted with higher concentrations. Similarly, the drug markedly impaired TG secretion into the medium. On the basis of these combined data, we were able to conclude that this cdcium antagonist impairs fat absorption at the cellular level, inhibiting TG and chylomicron secretion. These findings hfiermore suggest that intracellular cdciurn is necessary for normal intestinal lipid processing. In another study (Levy et a%.1 9 9 1 ~ we ) ~ demonstrated that the edcium channel blocker is a potential antiatherosclerotic agent capable of decreasing plasma lipids %ara atherogenic lipoproteins as well as aortic fatty streaks. Therefore, its effects are not exclusive to the intestine.

Ontogeny of imtestfml lipopratdn synthesis Data are generally lacking regarding the ability of the developing intestine to synthesize and secrete lipoproteins. Whereas a substantid body of literature covers gastrointestinal development, including moqhology and various digestive functions, no focus has k e n placed on the ontogeny of intestinal lipoproteins. By I1 weeks of gestation, the entire small intestinal mucosa is already limed by villi, which reveal a layer of epithelial cells (MCnard 1989). By 16 weeks, the overdl morphological appearance of the smdl intestine is very similar to that of the adult intestine (Mdnard 1989). During fetal life? humans develop disaccharidase and protease activities, seemingly in preparation for the digestion of glucose pBymers m d proteins at birth. Moreover, in most m from intra- to extra-uterine life includes an increase in lactase activity ((Kretchmer 1971) in preparation for the high lactose content of the milk (Jenmess et A. 1964). Other enzymes such as weurminidase (Dickson and Messer 1966) and a-glucosidase (Reddy and Wostmmn 19%) follow a similar ontogenic pattern. In contrast, details concerning lipid processing in utero are not known. Ht has been established that the newborn period is characterized by incomplete fat absorption, particularly in the Bow birth weight premature infant (Levy and Roy 1989). During the newborn period, insufficient mounts of pancreatic lipase are found (Leknthal and Lee 1981; Zoppi et d. 1972) and the profile of feed lipids reveals a significant percentage of urhydrolyzed TG (Watkns et al. 1974). Lipase derived from human milk, lingad, and gastric sources actively contributes to the hydrolysis of exogenous TG (Blackberg and Hernell 1981; Hamosh 1979; Levy et d. 1981). These extrapancreatic lipslytic emymes thus play an important role during the postnatal period, a time characterized by inadequate exocrine pancreatic function and a relatively high fat intake. Despite their active contribution, steatorrhea is a constant finding in the newborn period. The immature liver dscs undergoes qualitative and qmntita-

414

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/11/14 For personal use only.

LEVY

tive changes in bile salt metabolism, which contribute to the mdabsoqtion in the periwatd period (Poley et al. 1964; W a t b s et d. 1973). The deficiency of bile sdt p o l and eoncentration is aggravated by immaturity of the active iled tramsport mechanism (Little and Lester 1980). Thus, the two primary intraluminal factors involved in fat absorption, lipolysis and bile acid induced micelle formation, are deficient in the low birth weight infant and newborn. However, the cellular phase of fat absorption, which includes the processing of lipids by fetal or newborn enterocytes and their transport by lipoproteins, is an equally important, but largely unknown step in this complex process. We have recently carried out studies to explore the ontogeny and site of intestinal lipid md lipoprotein synthesis in rats (Mehran et d. 1992). In this study, the incopration of oleic acid was higher for the fetal explmts. However, the efficiency of esterifimtion of free fatty acids into triglycerides in the jejunum increases with age. The same profile was found at the ileal site for the incorporation of oleic acid, but the capacity for the synthesis of triglycerides was more intense during the sucHing period. We also demonstrated that fetal explants are able to synthesize the three types of lipoproteins, chylomicroms, VEDE, and HDE, as early as 18 - 19 days of gestation. Thus, we found no evidence for immaturity of the intestinal cells to package and transport dietary lipids at this stage of gestation. The differences mong both the different age groups ( f e d , suckling, weaning) md the two intestinal sites can be attributed to regulatory factors present during the development. These could be the intrinsic factors that control cellular maturation md hnctioning, and extrinsic factors such as dietary variations. Obviously, there are limitations in evaluating intestinal lipoprotein secretion using a rat model, and difficulty in extrapolating the data to higher rn als. We have therefore performed other experiments to investigate intestind lipprotein synthesis and secretion in the humm fetus (Levy et d. 1991a). This experimental approach was quite pertinent given that (I) a variety of lipids occurs in amistic fluid and lipid concentrations increase as gestation progresses; (2) swallowing begins as early as I1 weeks before suckling behavior (Herbst 1989), and lipids are present in meconium detected in the lumen of the intestine (Bernhard and Lindlar 1956); (3) by the 10 - 12th week of gestation, villi have appeared, mature junctional complexes are present, the basement lamina is well developed, microvilli contain well-formed glycocalyx, a d rnitochondrial md Golgi Iwdizations are the same as in fully developed epithelial cells (MCnard 1989); md (4) contrary to our meager howledge of developmewtd lipoproteins, several reports have f i d y stressed the intestinal synthesis of human approteins in human fetuses (Zmnis et al. 1980) and suckling a n i d s (Black and Davidson 1989). These findings may reflect the pssibility of active lipoprotein synthesis and secretion by the fetal intestine. Using jejunal explants incubated with [""C]oleate, we have recently demonstrated that human fetid intestine has the capacity to elaborate d l four classes of lipoproteins: chylomicrsns, VLDL, LDE, and HDL (Levy et d. 19914. Although chylomicrons were the predominant particles to carry newly labelled lipids, d B lipoprotein fractions were TG-enriched. From the preceding discussion, it is clear that the rnucssd phase of lipid absorption is intact in developing intestine. The intracellular metabolic mechanisms required for lipoprotein production are already present in jejunal epithelial cells

between 17 and 20 weeks of gestation. However, it is evident that other probes need to be used to examine the regulatory mechanisms of the various processes. While modulation of brush border enzymes a d epithelial cell proliferation by various regulatory factors was extensively investigated, no studies have been carried out on mabratiorad alterations in the intestinal formation and transport sf lipoproteins during development. Recently, we have attempted to examine the effect of epidermal growth factor (EGF). The organ culture technique, which is known to isolate the intestine from various influences, was utilized. The addition of EGF to the culture medium (25, 50, and 100 pg/mL) significantly enhanced chylomicron secretisn and decreased VLDL output into the medium (Levy et d m1991s). This novel finding following EGF administration is compatible with Tso's reports suggesting that the pathways for the formation of chylomicrons md VLDL are different (Tso et al. 1984) An alternative explanation is that intestind VLDL expand to form chylomicrons. T h s , these experimental manipulations are an excellent tool by which information in this area could be gained.

Disorders of lipid metabolism constitute a wide variety of important pathological conditions that affect persons sf d l ages. Especially, premature and newborn children are faced with considerable nutritional requirements for growth and development. Since dietary lipids serve as a vital source of energy and as an essential component of cell membranes, the demands on the mechanisms for fat absorption are high early in life. A sound background in developmental aspects of lipoproteins and a better understanding of the factors that influence and regulate their synthesis, assembly, and delivery will likely Bead to a more rational approach to the i absorptive apparatus, which characterizes the premature and newborn. In addition, new therapeutic choices for a number of congenital and acquired conditions limiting fat absorption may ernamate from the research in this field, employing dietary or perhaps phzagmacologicd modulation of intestid lipoprotein synthesis and secretion. Bernhard, K.,and Lindlar?F. 1956. Uber die Lipid des Mekoniums. Helv. Chir. Acta, 39: 1443- 14-45. Black, D. B., and Davidson, N. 0. 1989. Intestinal apolipoprotein synthesis and secretion in the saackJing pig. 3. Lipid Res. 36):207 218.

Blackberg, L., and Hemell, 0.1981. The bile salt seimu%at&lipse in human milk. Purification and characterization. Eur . J . Bicachem. 116: 221-225.

Brasitus, T. A., Davidson, N. O . , and Schachter, D. 1985. Variations in dietary triacylgycerol saturation alter the lipid cornpsition and fluidity of rat intestinal plasma membranes. Biochirn. BEophys. Acta, $12: 460-472. Cardell, R.R., Jr., Badenhausen, S., and Porter, K. R. 1967. Intestind triglyceride absorption in &herat. An electron rnicrssseopical study. 4. Cell. Biol. 34: 123-155. Chen, S. H., Habib, G . , Ymg, C. Y., Gu, Z. W . , Lee, B. R., Weng, S. A., Silvemn, S. R., Cai, S. J., Deslypere, J . P., Rosseneu, M., Gotto, A. M., Jr., Li, W. H., and Chn, L. 1987. Apolipprotein B-48 is the product of a messenger RNA with an organ-specific in-frame stop codon, Science (Washingtow, D.C.), 238: 363 -366. Christon, W., Even, V., Davelmse, D., Lbger, C. L., and %Pipet9 J. 1989. Modification of fluidity and lipid -protein relationships in pig intestinal brush-border membrane by dietary essential fatty acid deficiency. Bischh. Biophys. Acta, 980: 77 -84.

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/11/14 For personal use only.

418

CAN. J . PHYSIOL. PHARMACBL. VOL. 40, 1992

Danid, H. B., Neugehuer, Be,Kratz, A., and Rehmer, 43. 1985. Eodkation of acid microclimate along intestinal villi of rat jejunum. Am. J. Physisl. 248: G293-G298. Daiidson, N. O . , Grlos, a. C., and Lukaszewica, A. M. 1m. Apolipoprotein B mRNA editing is mdulated by thyroid ksmone andogs but not growth administration in the rat. Mol. Bndocrinol. 4: 779 -785. Dickson, J. J., and Messer, M. 1966. Intestinal neuraminidase activity of suckling rats and other m m a l s : relationship to sialic acid content of milk. Arch. Biwhern. Biophys. 113: 609 -616. Field, F. J. 1984. Intestinal cholesterol esterase: intracellular emyme conhmimtion of cytosol by pancreatic enzymes. J. Lipid Wes. 25: 103- 109. Glatz, J. F. C., and Vwrhmp, J, M. 1985. Intracellular fatty acid binding proteins. Int. J . Bischem. 17: 13-22. Glicban, W. M.,Rogers, M., and G l i c b w , J. N. 1986. Apolipoprotein B synthesis by human liver and intestine in vitro. Prm. Nad. Acad. Sci. U.S.A. 83: 5296-5360. Goldstein, J. L., and Brown, M. S. 19'64. The low density lipoprotein pathway and its relation to atherosclerosis. Annu. Rev. Biochern. 4-6:897 -930. Gasrgstrsrn, B., and Erlanssn, C. 1941. Pancreatic juice co-lipase: physiological importance. Biochirn. Biophys. Acts, 242: 509 5 13. Green, P. H. R., and Glickman, R. M. 1981. Intestinal lipoprotein rne%abolisrn. S. Lipid Res. 22: 1153. Green, P. R. R.,Tall, A. R., and Glichan, 8. M. 1978. Rat intestine secretes discoid high density lipoproteins. J . CHin. Invest. 61: 528-534. Hamosh, M. 1979. A review. Fat digestion in the newborn: role of lingual lipase and predudenal digestion. Pediatr. Res. 13: 615 622. Hansen, A, Be,Wiese, H.F., Bodesche, A. N.,Haggard, M. E., Adams, J . D., and Davis, H. 1963. Role of liaoleic acid in infant nutrition. Pediatrics, 31: 171-191. Haung, R.,and Nomm, K. R. 1976. Coenzyme A dependent esterification sf cholesterol in rat intestinal mucosa. Scad. J. Gastroenkerol. 11: 615-621. Herbert, PaN., Assmann, G., Gotto, A. M., Jr., and Fredrickson, D. S. 1983. F a d i a l lipoprotein deficiency: abetalipoproteinemia, Hny~b~ipopsoteinernia, and Tangier disease. 1' The metabolic basis of inherited disease. Edited by J. B. Stanbury, J. B. Wyngauden, D. S. Fredrickson, J. L. Goldstein, and M. S. Brown. McGraw-Hill, New Y s k . pp. 589-621. Werbst, J. J. 1989. Development of suck and swallow. Bm Human gastrointestinal development. Edited by E. Lebenthh. Raven Press, New York. pp. 229-239. Hjelte, L., Melin, T., Nilsson, A., and Strandvik, B. 1990. Absorption and metabslism of [3H]arachidsnicand [i4C]linolenicacid in essential fatty acid-deficient rats. Am. J. Bhysiol. 259: G1 16G124. Holman, W. T. 1968. Essential fatty acid deficiency. Prog Chem. 9: 279 -348. Hospattmkar, A. V . , Hipchi, K., Law, S. W., Meglin, N., and Brewer, H.B. 1984. Identification of a novel in-frame translational stop codon in human intestine apo B mRNA. Biochern. Biophys. Res. C o m u n . 14.8: 279-285. Jenness, R., Regehr, E. A., and Sloan, W. E. 1964. Comparative biochemical studies of milks. I1 Dialyzable carbohydrates. Gomp. Biochern. Physiol. 13: 339-352. Johnston, J. M. 1978. Esterification reactions in the intestinal mumsa and lipid absorption. In Disturbances in lipid and lipoprotein metabolism. Edited by J. M. Dietschy, A. M. Gotto, Jr., and b. A. Ontko. American Physiological Society, Bethesda, Maryland. pp. 57-68. Kane, 3. P. 1983. Apslipsprotein B: structural and metabolic heterogeneity. Amu. Rev. Physiol. 45: 637 -650. Kretchmer, N. 1971. Lactose and lac~ease- a historical perspective. Gastroenterology, 61: 805 -8 13. kbenthal, E., and Lee, P. C. 1981. The development of pancreatic

function in premature infants after milk-based and soy-baxd formulas. Pediatr. Res. 15: 1240-1244. Leblond, C. P. 1981. The life story of cells in renewing systems. Am. J. Anat. 160: 114-158. Lepage, G., Levy, E., Ronco, N., Smith, L., Galeano, N, and Roy, C.C. 1989. Direct tramesterification of plasma fatty acids for the diagnosis of essential fatty acid deficiency in cystic fibrosis. J. Lipid Res. 36: 1483- 1490. Levy, E., and Roy, C. C. 1989. DevelopmewtaI aspects of intestinal lipoprotein synthesis and secrdion. IB H u m n gastrointestinal development. Edited by E. kbenhal. Raven Press, New York. pp. 491 -502. Levy, E., Goldstein, R., Freier, S . , and Shaf-ir, E. 1981. Characterization of gastric ligslytic activity. Biochim. Biophys. Acta, 50: 183- 190. Levy, E., Goldstein, R., Freier, S., and Shafiir, E. 198%.Gastric lipase in the newborn rat. Pediatr. Res. 16: 69 -74. Levy, E., Gddstein, R., Sta&iewicz, H.,Hager, E., Faber, J., and Freier, S . 1984. Gastric handling of medium chain triglycerides and subsequent me&klisrn in the suckling rat. J. Pediatr. Gasfroenteml. Nutr. 3: 784 -789. Levy, E., Marcel, Y. L., Milne, R. W., Grey, V. L., and Roy, C. C. 1987a. Absence of intestinal synthesis of aplipoprotein B 4 8 in two cases of snbekdipsproteinemia. Gastroenterology, 93: 1119-1126. Levy, E.,Marcel, Y., Deckelbaurn, 8. J., Mike, R., Ispage, G., Seidman, E., Bewdayan, M., md Roy, C. C. 1987b. Intestinal a p B synthesis, lipid, md lipoproteins in chylomicron retention disease. 3. Lipid Res. 28: I263 - 1274. Levy, E.,Chouraqaai, J. P., and Roy, C. C. 1988. Steatorrhea and disorders of chylomicron synthesis and secretion. Bdiatr. Clin. North Am. 35: 54 -66. Levy, E., Lepage, Go, Bendayan, M., Ronco, N., Thibaolt, L., Galearno, N., Smith. L., and Roy, @. C. 1989. Relationship of decreased hepatic lipase activity and lipoprotein abnomlities to essential fatty acid deficiency in cystic fibrosis patients. J. Lipid Res. a@:1197-1209. Levy, E., Thibault, L., Garofdo, C., Messier, M.,Lepage, G . , Ronco, N., and Roy, C. C. l9Wa. Combined (n-3 and m-6) essential fatty acid deficiency is a potent mdarlator sf plasm lipids, lipprotein composition, and lipolytic enzymes. 3. Lipid Res. 31: 2009-2017. Levy, E., Rochette, C . , Losadom, I., Roy, C. C.,Milme, R. W., Marcel, Y. L., and Bendayan, M. 1SWb. Agolipoprotein B- 100: Imunolocdization and synthesis in human intestinal mucosa. J. Lipid Res. 31: 1934-1946. Levy, E., Thibsnult, L., and Menard, D. I991a. Intestinal lipoproteins in the human fetus: modulation by epidermal growth factor (EGF). J. Lipid Res. In press. Levy, E.,Tardif, J., Russo, B., Lavigne, F., Thibault, L., St-Louis, I., Garofalo, C., Benhyan, M., Bouthillier, D., and Garceau, D. 1991c. Effect of clentimem on lipid profile, lipoprotein mmpsition and aortic fatty streaks in cholesterol-f& rabbits. Atherosclerosis 90: 141- 148. Levy, E., Wouleau, T., Lepage, G., Smith, L., Junzien, J. I&., and Roy, C. C. 199Id. Partially purified rabbit gastric lipase: In vitro and in vlvo experiments to assess its potentid contribution to gastric and intestinal liplysis. Nutr. Res. 11: 607-619. Levy, E., Garofals, C., Thibault, E., Disnne, S., Daoust, L., kpage, G., and Roy, G. G. 1992a. Intraludnal and intracellular phases of fat absorption are impaired in essential fatty acid deficiency. Am. J. Physisl. 262: G319-G326. Levy, E., Smith, L., Dumont, E., Garcau, D., Garofdo, C., ThiBnlt, L., and Seichan, E. 1992b. The effect of a new calcium channel blocker (TA-3880) on lipoprotein profile and intestinal lipid handing in rodents. Prm. Soc. Exp. Biol. Md. 199: 128- 135. Little, %.M., and Lester, R. 1980. Ontogenesis of intestinal bile salt absorption in the neonatal rat- Am. J. Physiol. 227: 390-395. Mahley, R. W., Innemrity, %. L., Rall, S . C . , and Weisgraber,

LEVY

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/11/14 For personal use only.

M. H. 1984. Plasm lipoproteins: aplipoprotein structure and function. J. Lipid Res. 25: I277 - 1294. Mehran, M., Thibault, L., Russo, P., Garofdo, C., and Levy, E. 1991. The ontogeny and site of intestind lipid and lipprotein synthesis. Eur. J. Clin. Invest. In press. Memrd , D . 1989. Growb -promoting factors and the development of the human gut. Pn H u m n gastrointestinal development. Edited by E. Lebenhd. Raven Press, New York. pp. 123- 150. Ney, D. M.,Ziboh, V. A., and Schneemn, B. 0. 1987. Reduction in plasm apo E and HDE levels in rats with essential fatty acid deficiency. J. Nutr. 11'8: 2016 -2020. Ocher, R. K., and Manning, J. A. 1974~.Intestinal fatty acid binding protein (FABP); studies on physiological function. b. Clin. Invest. 53: 53-57a. Ocher, R. K., and Manning, J. A. 1974b. Fatty acid-binding protein in small intestine. Identification, isolation and evidence for its role in cellular fatty acid transport. B. Clin. Invest. 54: 326-338. Ocher, R. K., Manning, J. M., Poppahausen, R. B., md Ho, W. K. k. 1972. A binding protein for fatty acids in cystosol of intestind mucosa, liver, myocardium, and other tissues. Science (Washington, DC), 177: 56 -58. Pastan, I., and WiB1ingham, M. C. 1985. The pathway of endscyto%is.In Endscytosis. Edited by I. Pastan and M. C. Willingham. Plenum Press, New York. pp. 1-44. Poley, J. R., Dower, J. C., Owen, C. A., Jr., and Stickler, G. B. 1964. Bile acids in infants and children. J. Lab. Clin. Med. 63: 838 - 846. Powell, L. Mha., Wallis, S. C., Pease, R. J . , Edwards, Y. H.,Knott, T. J . , and Scott, J. 8987. A novel form of tissue-specific RNA processing produces apolipoprotein B-48 in intesthe. Cell, 50: 831 -M0* Ramussen, H. 1986. The calcium messenger system. N. Engl. J. Med. 314: 109-4 - 1101. Rddy, B. S., and Wostmann, B. S . 1966. Intestinal disaccharidase activities in the growing gem-free and conventional rat. Arch. Biochem. Biophys. 113: 609 -616. Redgrave, IF. G. 1983. F o m t i o n and metabolism of chylornicrons. Rev. Physiol. 28: 183- 138. Roy, C. C., Weber, A. M., Lepage, G . , Smith, L.,and Levy, E. 1988. Digestive and absorptive phase anomlies associated with the exocrine pancretic insufficiency of cystic fibrosis. J . Pediatr. Gastroenterol. Nutr. '8: S 1-S7. Sabesin, S. M. 1976. Ulmstmctural aspects of the intracellular assembly, transport, and exocytosis of chylomicrons by rat intestinal absorptive cells. In Lipid absorption: biochemical and clinical aspects. Edited by K. R o m e l and H. Goebbel. University Press, Baltimore. p. 113.

419

Sabesin, S. M.,and Frase, S. 1977. Electron micmscopic studies of the assembly, intracellular transport, and secretion of chylomicrons by rat intestine. 9. Lipid Res. 18: 496. Schwartz, C. C., Halloran, L. G . , Vlahcevic, Z. R., Gregory, D. H., and Swell, L. 1978. Preferential utilization of free cholesterol for biliary cholesterol secretion in man. Science (Washington, DC), 2W:62 -64. Shiau, Y. F., Femandez, P., Jackson, M. J., and McMonagle, S. 1985. Mechanisms maintaining a low pH microclimte in the intestine. Am. J. Physiol. 248: Gm8-6617. Thornon, A. B. R., and Dietschy, J. M. 1981. Intestinal lipid absorption: major extracellular and intracellular events. In Physiology of the gastrointestinal tract. Edit$ by L. R. Johnson. Raven Press, New York. pp. 1 147- 1220. Tso, P., and GoUamudi, S. R. 1984. Pluronic L-8 1: a potent imhibotor of the transport of intestinal chylomierons. Am. J. Physiol. 24'8: G32 -636. Tso, P., Drake, D. S., Black, D. D., and Sabesin, S. M. 19846. Evidence for separate pathways sf chylomicron and very low density l i p r o t e i n assembly and transport by rat small intestine. Am. J. Physiol. 247: G599 -G610. Tytgat, G. N., Rubin, C. E., and Saunders, D. R. 1971. Synthesis and transport of lipoprotein particles by intestinal absorptive cells in man. J. Clin. Invest. 90: 2065 -2078. Watkns, 3. B., Szczepanik, P., Gould, J., mein, P. D.,and Lester, R. 1973. Bile salt kinetics in premature infants: an explanation for inefficient Iipid absorption Gastroenterology, 64: 817. (Abstr .I Watkins, J. B., Bliss, C. M., DonaHdssn, R. M.,and Lester, R. 1974. Characterization of newborn fecd lipids, Pediatrics, 53: 511 -515. Westergmd, H.,and Dietschy,J. M.1976. The mechanism whereby bile acid micelles increase the rate of fatty acid and cholesterol uptake into the intestinal mucosal cell. J . Clin. Invest. 58: 97. Windmueller, I%. G., and Levy, W. I. 1968. Production of @-lipoproteinsby the intestine in the intestine in the rat. J. Biol. Chem. 243: 4878-4884. Yurkowslci, M., and Walker, B. L. 1970. Lipids of the intestinal mucosa of n o m l and essential fatty acid deficient rats. Can. J . Physiol. Phamcol. 48: 631 -639. Zannis, $I. I., Breslow, J. Lo,and Katz, A. J. 1988. Isoproteins of human apolipoprotein A-I demonstrated in plasma and intestinal organ culture. J. Biol. Chem. 255: 8612-8617. Zoppi, G . , Andreotti, G . , and Pajno-Ferra, F. 1972. Exocrine pancreas function in premature and full tern neonates. Bediatr. Res. 6: 880-886.