DEVELOPMENTAL
57, 1-14
BIOLOGY
(19771
Lipid Absorption MILLIE Department Received
Rat Intestine
I. FERLATTE AND FRANCES J. ZEMAN
of Nutrition, April
in Newborn
8, 1976;
University nrrepted
of California, in revised
Davis, form
California
December
95616
20,1976
There appear to be three distinct regions of cellular maturation within the newborn intestinal epithelium. As crypt cells move onto the lower villus surface, they become transition cells which in turn differentiate into mature villus-absorptive cells. In order to study lipid absorption in cells of each region, proximal segments of intestines of both suckled and corn oilinfused newborn rats were prepared by routine light and electron microscopic methods. Crypt cells were capable of division and, perhaps, of some absorption, whereas villus cells were capable only of absorption. Transition cells were capable of both division and lipid absorption. INTRODUCTION
investigated the morphological and histo chemical characteristics and functional capabilities of transition cells. They appear to be morphologically and functionally distinct from both villus and crypt cells, and they do not seem to correspond to any adult intestinal epithelial cells. This paper describes lipid absorption by intestinal cells from the newborn proximal small intestine and characterizes the function and morphology of transition cells.
Intestinal absorption processes have been shown to differ in neonatal and adult rats (Halliday, 1955; Brambell, 1958). Although there have been many investigations of lipid absorption in adult rat intestine (Palay and Karlin, 1959; Rostgaard and Barrnett, 1965; Cardell et al., 1967; Jersild and Clayton, 1971), and intestinal absorption of various substances has been studied in neonatal and suckling rats (Clark, 1959; Vacek et aZ., 1962; Koldovsky et al., 1963; Graney, 1968; Cornell and Padykula, 1969; Rodewald, 1970, 1973), few have examined lipid absorption in young rats (Vacek et al., 1962; Koldovsky et al., 1963; Cornell and Padykula, 1969). Since the diet of suckling rats is rich in lipid, and since lipid absorption in the mammalian tract occurs primarily within the proximal end of the small intestine (Borgstrom et al., 1957; Johnston, 1959; Dawson and Isselbacher, 1960), we investigated lipid absorption within the proximal intestine of newborn rats. Within the proximal intestinal epithelium of newborn rats, there appears to be a region composed of cells that are in transition from crypt to villus cells. These transition cells are located above the crypts along the lower edges of the villi. We have Copyright All rights
0 1977 by Academic Press, of reproduction in any form
Inc. reserved.
MATERIALS
AND
METHODS
Newborn rats of the Sprague-Dawley strain, weighing 6.4 * 0.3 g were used. Three groups were studied: unsuckled animals, suckled animals, and unsuckled animals infused with corn oil. The proximal small intestines of unsuckled animals were fixed in Bouin’s solution and prepared for light microscopy according to routine procedures. Paraffin sections were cut at 6 pm and stained with hematoxylin and eosin. To differentiate transition cells from crypt-top cells we histochemically analyzed epithelial cells for the presence of alkaline phosphatase, since, in newborn rats, the upper boundaries of the crypts are poorly defined. The proximal intestines of unsuckled animals were frozen in isopentane, chilled in liquid
1
ISSN
0012-1606
2
DEVELOPMENTAL
BIOLOGY
nitrogen, and sectioned at 10 pm. The sections were incubated for 1 hr at room temperature using naphthol AS-MX phosphoric acid as the substrate and red violet LB salt as the chromogen (Pearse, 1961). The sections were counterstained with methyl green and examined the same day. Pieces, 1-2 mm long, were excised from the proximal small intestine of suckled animals about 2 cm from the pyloric sphincter and were fixed in 2.7% glutaraldehyde buffered with 0.13 M sodium cacodylate, pH 7.4, for 1 hr at room temperature. Tissues were postfixed in 1% osmium tetroxide and 0.1 M sodium cacodylate for 1 hr at 4”C, rinsed in buffer, dehydrated through an ethanol series to propylene oxide, and embedded in Epon. Thick sections (1.5 pm) were stained with methylene blue and azure II. Thin sections were stained with uranyl acetate and lead citrate or uranyl acetate alone and were examined with Zeiss 9 and RCA-EMU 3G electron microscopes. Unsuckled newborn animals were anesthetized with sodium pentobarbital, and 0.1 ml of corn oil was infused into the small intestine via a cannula through the stomach and pyloric sphincter. Animals were sacrificed 1 or 3 hr later, and the intestinal tissue was processed as just described. RESULTS
Light
Microscopy
Crypt cells in newborn rats had basophilic cytoplasm and small intensely stained nuclei. A brush border was not evident. Mitoses were frequently observed within the crypts. Cells within the crypts did not reveal a positive response for alkaline phosphatase. On the other hand, intestinal absorptive cells in newborn rats were tall columnar cells with wide brush borders and large rounded nuclei. The apical portions of villus cells showed a positive response for alkaline phosphatase. Between these two types of cells, an intermediate type was observed. We have
b-OLUME
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1977
chosen to call these lower villus cells “transition cells”, since they appear to be a distinct cell type that occurs during differentiation in newborn rat intestinal epithelium. These epithelial cells, located along the lower portion of the villus, were tall with elongated nuclei. The cytoplasm was composed of basophilic and acidophilic areas, and the cells had a narrow brush border. These cells gave a positive reaction for alkaline phosphatase on their apical surfaces, distinguishing them from crypt cells. There were numerous instances of mitosis in these cells (Fig. 11, indicating that they were capable of division. After suckling, a few crypt cells were observed to contain osmiophilic droplets. Droplets were rarely visible within mature absorptive cells, and, in the rare instance when droplets were observed, they were generally in cells at the villus tips. Transition cells contained many large osmiophilic droplets distributed throughout the cytoplasm (Fig. 2). Figures 3 and 4 show a transition cell undergoing mitosis in which the cytoplasm contains several droplets. The osmiophilic droplets appeared to be lipid, but since the presence of lipid within cells along the lower villus surface had not been previously described, it was necessary to determine if the transition cells would absorb lipid. One hour after infusion of corn oil, there were no large lipid droplets within crypt or transition cells. However, following a 3-hr exposure to corn oil, we observed many lipid droplets within transition cells similar to those seen in transition cells from suckled animals. Electron
Microscopy
Crypts in newborn rat intestines were not well developed, and their upper limits were not well defined. Crypt cells had sparse stubby microvilli, numerous free riand mitochondria with less bosomes, rough or smooth endoplasmic reticulum than that found in villus cells (Fig. 5).
FERLATTE
AND ZEMAN
Absorption
FIG. 1. Light micrograph of villi of unsuckled animal. The top of the crypt is marked with an arrow. A transition cell is undergoing mitosis (M). Hematoxylin and eosin. x480.
After a l-hr exposure to corn oil, there were no large lipid droplets observed within crypt cells, but, after a prolonged exposure to lipid, some crypt cells contained lipid droplets. Cells undergoing mitosis were observed to contain large 1.5 pm-diameter lipid droplets. Pinocytosis of lipid was not observed in any crypt cells, though small lipid droplets, some unbound and others contained within apical cisternae, were observed near the cell surface. Large unbound lipid droplets were formed by coalescence of small lipid droplets. Small lipid droplets were observed within Golgi vacuoles, but saccules remained flattened. Some chylomicra were observed within extracellular spaces, but were far less numerous than were the chylomicra observed within the extracellular space between villus cells.
in Newborn
Rat Intestine
3
FIG. 2. Light micrograph of villi of suckled animal. Transition cells contain numerous lipid droplets (L) shown here as dark inclusions. Only a few absorptive cells appear to contain lipid. Cells deep in the crypts (Cl are basophilic and do not contain lipid. Methylene blue and azure II. x 150.
With low power electron microscopy, the brush border of mature absorptive cells was observed to be composed of long narrow microvilli. Although microfilaments extended from the microvilli into the underlying cytoplasm, there were no perpendicular connecting microfilaments to form a well defined terminal web (Fig. 6). The lateral cell membranes were closely apposed and convoluted. Following suckling, the cells contained large numbers of small lipid droplets, predominantly in the supranuclear cytoplasm. Golgi bodies were composed of enlarged saccules containing numerous chylomicra. Occasionally, chylomicra were seen within the supranuclear intercellular space; however, most of the intercellular lipid was below the level of the nuclei. After suckling or after corn oil infusion, there were large lipid droplets in the intestinal lumen. Luminal lipid was surrounded by a light halo covered with an
DEVELOPMENTAL
BIOLOGY
FIG. 3. Light micrograph of suckled animal. Transition cells contain numerous osmiophilic lipid droplets. Crypt cells (0 do not contain lipid droplets. Due to an oblique cut, the intestinal lumen does not extend to the base of the crypt. A transition cell containing lipid is undergoing mitosis (Ml. Methylene blue and azure II. x 375. FIG. 4. Higher power micrograph of the mitotic transition cell (M) in Fig. 3. x 600.
osmiophilic rim. Where the lipid was in contact with microvilli, the halo and rim appeared disrupted, and the lipid outline lost its spherical quality (Fig. 7). The microvilli did not appear to come into direct contact with the lipid droplet; rather, they were connected by fibrils composing the cell coat. Pinocytotic vesicles were few and did not appear to contain lipid. Smooth apical vesicles were observed to contain lipid droplets. Deeper in the cytoplasm, rough endoplasmic reticulum (RER) was often observed to contain lipid droplets (Fig. 8). Mitochondria were numerous and closely surrounded by RER. Some lipid droplets were covered with or surrounded by fine electron-dense parti-
VOLUME
57, 1977
cles. These particles were due to the lead stain, as they were not present when the lead stain was omitted. Not all lipid droplets reacted with the lead to the same degree; while some droplets were sharply delimited by numerous particles, others had only a few particles overlying them or no depositions. Lipid droplets that were enclosed within endoplasmic reticulum were not observed to have a heavy lead deposition. Unbound droplets were densely surrounded by the particles, and, perhaps, the surrounding membrane inhibited a heavy deposition of lead about the periphery. Golgi bodies had enlarged vacuoles containing chylomicra, still delimited by lead particles (Fig. 9). After chylomicra were transported to the extracellular space, they did not accumulate the electron-dense particles about their peripheries: The luminal surfaces of transition cells were composed of numerous short microvilli, intermediate in length between those of villus and crypt cells and covered with a fuzzy layer. Lateral cell membranes were closely apposed and connected at the apical end by junctional complexes. Adjacent cells exhibited some lateral interdigitations, whereas some of the lateral cell membranes appeared straight. The terminal web was not well formed. Smooth vesicles were present in the apical portion of the cell, as well as dense bodies and multivesicular bodies. Mitochondria and numerous free ribosomes were seen throughout the cell, but free ribosomes were less numerous than in crypt cells. RER, generally in close conjunction with mitochondria, was more abundant than in crypt cells. Electron microscopy confirmed that, after 1 hr of exposure to corn oil, there was no appreciable amount of absorbed lipid within transition cells, but, after a 3-hr exposure to the oil, transition cells were filled with numerous lipid droplets of various sizes (Fig. 10). Some of the droplets were membrane-bound and others were free in the cytoplasm. Large lipid droplets
FERLATTE
AND ZEMAN
FIG. 5. Crypt cells from suckled newborn Golgi apparatus (G) is composed of flattened present. x 17,760.
Absorption
in Newborn
Rat Intestine
contain numerous mitochondria saccules and small vacuoles.
were formed by coalescing smaller ones. Cellular organelles were displaced by enormous lipid droplets (Figs. 10 and 111. A few small droplets were observed within the cell adjacent to the apical cell surface. Although pinocytotic vesicles were observed, none appeared to contain lipid. Lipid droplets were often in close conjunction with mitochondria and RER. Some of the droplets were contained within the RER. The Golgi apparatus was active, as indicated by enlarged vacuoles, but not to the same extent as in mature absorptive cells. Extracellular lipid which had been transported through the cell was present as a few discrete droplets or as droplets so numerous that the entire extracellular space was occluded.
and free ribosomes A few lipid droplets
5
(RI. The (L) are
DISCUSSION
Currently, two methods of lipid absorption in the small intestine have been proposed, passive diffusion of hydrolyzed lipid and bulk pinocytosis of lipid. Briefly, passive diffusion of lipid occurs as follows. Intestinal luminal content consists of an oily phase and a micellar phase (Hofmann and Borgstrom, 19621. Components of the oily phase, triglycerides, are transformed into a micellar solution of monoglycerides and free fatty acids by the action of pancreatic lipase and bile salts. Monoglycerides and free fatty acids then passively diffuse through the plasma membrane (Johnston and Borgstrom, 1964; Jersild, 1966; Cardell et al., 1967; Johnston, 19681, where they are resynthesized into triglyc-
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DEVELOPMENTAL
BIOLOGY
VOLUME
FIG. 6. Low power electron micrograph of villus-absorptive extend into the cytoplasm, but a well-formed terminal web bound lipid droplets (L) are present. x 17,760.
erides within the SER (Cardell et al ., 1967; Strauss, 1968). Although pinocytosis of lipid has been reported by some investigators (Palay and Karlin, 1959; Ashworth et al., 1960; Cornell and Padykula, 1969), others have not observed pinocytosis of lipid following ingestion of fat (Ladman et. al., 1963; Phelps et al., 1964; Cardell et al., 1967; Dermer, 1967; Oledzka-Slotwinska and
57, 1977
cells of a suckled animal. Microfilaments is not evident. Numerous small membrane-
Desmet, 1971). Consequently, pinocytosis has not been universally accepted as a major route of lipid absorption (Strauss, 1968). Senior (1964) has pointed out that the two methods of fat absorption, diffusion of micellar components with intracellular resynthesis and bulk pinocytosis of unhydrolyzed lipid, are not mutually exclusive. He suggested that, as large lipid droplets are
FERLATTE
AND ZEMAN
Absorption
hydrolyzed, smaller unhydrolyzed droplets are formed which may be engulfed in apical pits. However, we did not observe small lipid droplets near large luminal lipid droplets, and it has been shown that the apical pits are not involved in pinocytosis of lipid (Cardell et al., 1967; Rodewald, 1970). Since lipid is absorbed more rapidly in lo-day-old rats than in adults, it has been
in Newborn
Rat Intestine
7
proposed that fat absorption in suckling rats differs from that in adult animals (Koldovsky, 1969). It has been suggested that neonatal rats are able to absorb triglycerides intact without prior hydrolysis (Koldovsky et al., 1960). Cornell and Padykula (1969) observed pinocytosis of lipid within ileal cells of neonatal rats. In the newborn rats in this study, we did not observe pinocytosis of lipid nor were we
FIG. 7. A lipid droplet within the intestinal lumen is surrounded by a light halo (H) and an osmiophilic rim CR). Where it is in contact with the glycocalyx, the spherical outline breaks down (arrows). Small lipid droplets CL) are contained within smooth vesicles and unbound lipid KJL) is free in the cytoplasm of a villus absorptive cell. x 14,400.
8
DEVELOPMENTAL
FIG. 8. Columnar cell of suckled and mitochondria (M). Lipid droplets dense particles. x 32,600.
BIOLOGY
animal showing (L) are within
VOLUME
57, 1977
close apposition of rough endoplasmic reticulum (RER) the RER. Unbound lipid (UL) is surrounded by electron-
able to identify lipid within the terminal web area; we did so only deeper in the cytoplasm, suggesting that newborn duodenal cells absorb lipid by diffusion across the plasma membrane. Crypt cells, distinguishable from absorptive cells because of cytoplasmic basophilia and from villus cells by the presence of alkaline phosphatase (Padykula, 1962; Overton, 1965; Millington and Brown, 1967; Oledzka-Slotwinska and Desmet, 19711, were similar to those described by Trier and Rubin (1965). The cytoplasmic basophilia of crypt cells is due to a high concentration of RNA (Padykula, 1962) existing as free ribosomes, a characteristic of undifferentiated cells. After a long exposure to fat, only a few crypt ceils contained lipid, resulting possibly from absorption or from synthesis of lipid from simple precursors derived in part from the plasma. Build-up of lipid within immature intestinal cells has been attributed to storage for intracellular use (Dobbins, 1969). In the case of crypt cells, glycerolipid is necessary for the synthesis of membrane phospholipid for newly formed cells (Gang1
and Ockner, 1974). The ultrastructure of absorptive cells from the proximal small intestine of newborn rat pups appeared similar to that of previous descriptions of mature villus cells (Palay and Karlin, 1959; Dunn, 1967; Cardell et al., 1967; Strauss, 1968; Rodewald, 1973). Since the ultrastructure of lipid absorption has been described in detail (Cardell et al., 1967; Strauss, 1968; Cornell and Padykula, 19691, only those observations that add new information will be discussed here. The discussion will follow the path of lipid as it moves from the intestinal lumen through the cell to enter the extracellular space. Luminal lipid did not come into direct contact with the apical cell membrane, but was separated by the glycocalyx covering the surface of the microvilli. The glycocalyx may play a role in lipid absorption, as evidenced by the intimate relationship between the glycocalyx and lipid droplets. The outlines of luminal lipid droplets were round and smooth except where they were in contact with the glycocalyx, suggesting
FERLATTE
AND ZEMAN
Absorption
FIG. 9. An electron micrograph of a Golgi body (G) within an absorptive cell. The chylomicra within the Golgi are delimited by electron-dense particles. The chylomicra (CH) within the extracellular space (ES) do not aggregate lead particles. x 33,300.
that the glycocalyx may assist in the breakdown of lipid. The close relationship between RER and mitochondria has been previously reported (Cardell et al., 1967; Hayward, 1967; Friedman and Cardell, 1972). Smooth and rough ER have been shown to be in continuity in the adult animal, and lipid droplets have occasionally been observed within RER, but RER has not been considered a path-
in Newborn
Rat Intestine
9
way of lipid from SER (Cardell et al., 1967; Dobbins, 1969; Friedman and Cardell, 1972). Cardell et al. (1967) attributed the presence of lipid droplets within RER to its synthesis in adjacent SER. We observed numerous instances of lipid droplets within RER and feel that RER plays a role in the transport of lipid through the cell, perhaps in transporting the lipid from the SER to the Golgi body. Rough ER contributes the protein component of chylomicra (Dobbins, 19691, and it has been suggested previously (Senior, 1964) that the lipid droplet moves through the RER to pick up its protein coating. Electron-dense particles surrounding and covering the lipid droplet were apparently artifacts of lead staining as previously demonstrated (Rostgaard and Barrnett, 1965). However, it appeared that adjacent lipid droplets within the cell cytoplasm exhibited different affinities to the lead stain, perhaps due to differences in surface properties of the droplets. The differences in surface properties may be due to the presence of two types of intracellular lipid droplets, those bound within endoplasmic reticulum and unbound or matrix lipid (Friedman and Cardell, 1972). ERbound lipid did not appear to accumulate heavy lead deposits, whereas unbound lipid droplets were heavily stained by the lead. Although we could not discern a membrane about those droplets with a heavy deposition of lead, there exists the possibility that the lead deposition may have prevented our viewing it. The chylomicra within the Golgi body were outlined with dense particles, but, once the chylomicra had been transported from the cell, they were not. A carbohydrate moiety is added to chylomicra within the Golgi body (Friedman and Cardell, 1972), and this may alter their surface properties so the lead would not precipitate about their peripheries. The morphological appearance of lipid absorption within transition cells was unlike that within either crypt or absorptive
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DEVELOPMENTAL
BIOLOGY
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FIG. 10. A low power electron micrograph of transition cells from a suckled newborn. There are numerous lipid droplets (LI of various sizes throughout the cytoplasm and the extracellular space is filled with chylomicra (CH). x 6600.
FERLATTE
AND ZEMAN
Absorption
in Newborn
FIG. 11. Transition cells from suckled newborn animal with large lipid cytoplasm. The Golgi body (G), though small, contains chylomicra (arrow). ous than in crypt cells. x 11,571.
cells. Only in transition cells were there massive accumulations of lipid within the cells. It is possible that the transition cells were not absorbing lipid from the intestinal lumen, but rather were synthesizing it from simple precursors derived from the plasma as crypt cells do (Gang1 and Ockner, 1974). However, as transition cells will soon mature into absorptive cells, it
11
Rat Intestine
droplets within Free ribosomes
the supranuclear are less numer-
seems reasonable that the cells have the capability to absorb and transport lipid through the cells. However, transition cells appeared restricted in their capacity to transport lipid through the cell. Rough ER, the site of protein synthesis, was less abundant in transition cells than in absorptive cells, and the Golgi apparatus was less active as judged by fewer cristae and
12
DEVELOPMENTAL
BIOLOGY
smaller vacuoles. Both of these organelles have been shown to play important roles in the formation of chylomicra (Isselbather, 1965; Dobbins, 1969). Friedman and Cardell (1972) utilizing puromycin to block protein synthesis, produced absorptive cells filled with lipid, similar to transition cells of suckled rats. They proposed that the action of puromycin prevented the RER from synthesizing membranes destined for the Golgi apparatus, causing a reduction of Golgi membranes which normally envelop the chylomicra, and suggested that, without the enveloping membranes, the cells were unable to release the chylomicra, thus causing a buildup within the cells. Differentiation of transition cells has progressed to allow for some chylomiera formation, as evidenced by the large numbers of chylomicra within the extracellular space at the bases of the cells, but the cells have not acquired the mature complements of RER and Golgi apparatus that are present in absorptive cells. Progressive differentiation of epithelial cells occurs as they migrate from the crypt along the villus (Padykula, 1962; Lipkin, 1973; Cheng and Leblond, 1974). Transition cells in the newborn intestine appear to represent an intermediate cell type in the maturation process from crypt to villus. Transition cells, unlike crypt and villus cells, are capable of both absorption and division. The presence of mitotic figures within the intestinal epithelium has been described as occurring only within the confines of crypts (Leblond and Stevens, 1948; Cairnie et al., 1965; O’Connor, 1966; Wright et al., 1975). There does not seem to be an overlap of functional capabilities in adult intestinal epithelium, as crypt cell proliferative capability ceases prior to attainment of villus absorptive function. In most organ systems proliferation and differentiation are mutually exclusive, but, in some instances, a degree of overlap can be seen. In developing pancreas, mitotic cells contain zymogen (Wessells, 19641, and plasma cells can develop
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57. 1977
antibody before the cessation of cell proliferation (Makela and Nossal, 1962). Unlike adult intestinal cells which lose crypt mitotic function prior to gaining villus absorptive and transport functions, it appears that newborn intestinal cells retain their ability to divide while their capability to absorb and transport nutrients increases. This situation is advantageous for newborn animals with their shorter villi and shallow ‘crypts, as they are able to utilize all of their villus cells plus transition cells to some extent for absorption, and they have a proliferating pool of cells composed of both crypt and transition cells. This research was supported, in part, by U.S. Public Health Service Grant No. HD-06465 from the National Institute of Child Health and Human Development. REFERENCES C. T., STEMBRIDGE, V. A., and SANDERS, E. (1960). Lipid absorption, transport and hepatic assimilation studied with electron microscopy. Amer. J. Physiol. 198, 1326-1328. BORGSTR~M, B., DAHLQUIST, A., LUNDH, G., and SJOVALL, J. (1957). Studies of intestinal digestion and absorption in the human. J. Clin. Invest. 36, X21-1536. BRAMBELL, F. W. R. (1958). The passive immunity of the young mammal. Biol. Reu. 33, 488-531. CAIRNIE, A. B., LAMERTON, L. F., and STEEL, G. G. (1965). Cell proliferation studies in the intestinal epithelium ofthe rat. Exp. Cell Res. 39,528-538. CARDELL, R. R., JR., BADENHAUSEN, S., and PORTER, K. R. (1967). Intestinal triglyceride absorption in the rat. An electron microscopical study. J. Cell Biol. 34, 123-155. CHENG, H., and LEBLOND, C. P. (1974). Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. I. Columnar cell. Amer. J. Anat. 141, 461-479. CLARK, S. L., JR. (1959). The ingestion of proteins and colloidal materials by columnar absorptive cells of the small intestine in suckling rats and mice. J. Biophys. Biochem. Cytol. 5, 41-49. CORNELL, R., and PADYKULA, H. A. (1969). A cytological study of intestinal absorption in the suckling rat. Amer. J. Anat. 125, 291-316. DAWSON, A. M., and ISSELBACHER, K. J. (1960). The esterification of palmitate-l-‘4C by homogenates of intestinal mucosa. J. Clin. Inuest. 39, 150-160. DERMER, G. B. (1967). Ultrastructural changes in the microvillous plasma membrane during lipid
ASHWORTH,
FERLATTE
AND ZEMAN
A bsoi rption
absorption and the form of absorbed lipid: An in viva study. J. Ultrastruct. Res. 20, 51-71. DOBBINS, W. 0. III (1969). Morphologic aspects of lipid absorption. Amer. J. Clin. Nutr. 22,257-265. DUNN, J. S. (1967). The fine structure of the absorptive epithelial cells of the developing small intestine of the rat. J. Anat. (London) 101, 57-68. FRIEDMAN, H. I., and CARDELL, R. R., JR. (1972). Effects of puromycin on the structure of rat intestinal epithelial cells during fat absorption. J. Cell Biol. 52, 15-40. GANGL, A., and OCKNER, R. (1974). Intestinal metabolism of plasma free fatty acid. Clin. Res. 22, 358A. GRANEY, D. 0. (1968). The uptake of ferritin by ileal absorptive cells in suckling rats. An electron microscope study. Amer. J. Anat. 123, 227-254. HALLIDAY, R. (1955). The absorption of antibodies from immune sera by the gut of the young rat. Proc. Roy. Sot. B 143, 408-413. HAYWARD, A. F. (1967). Changes in fine structure of developing intestinal epithelium associated with pinocytosis. J. Anat. 102, 57-70. HOFMANN, A. F., and BORGSTRBM, B. (1962). Physice-chemical state of lipids in intestinal content during their digestion and absorption. Fed. Proc. 21, 43-50. ISSELBACHER, K. J. (1965). Metabolism and transport of lipid by intestinal mucosa. Fed. Proc. 24, 16-22. JERSILD, R. A., JR. (1966). A time sequence of fat absorption in the rat jejunum. Amer. J. Anat. 118, 135-162. JERSILD, R. A., JR., and CLAYTON, R. T. (1971). A comparison of the morphology of lipid absorption in the jejunum and ileum of the adult rat. Amer. J. Anat. 131, 481-503. JOHNSTON, J. M. (1959). Site offatty acid absorption. Proc. Sot. Exp. Biol. Med. 100, 669-670. JOHNSTON, J. M. (1968). Mechanism of fat absorption. In “Handbook of Physiology, Alimentary Canal” (C. F. Code and W. Heidel, eds.), Vol. 3, pp. 1353-1375. Williams and Wilkins, Baltimore. JOHNSTON, J. M., and BORGSTR~M, B. (1964). The intestinal absorption and metabolism of micellar solutions of lipids. Biochem. Biophys. Acta 84, 412-423. KOLDOVSKY, 0. (1969). “Development of the Functions of the Small Intestine in Mammals and Man,” p. 85. Karger, Basel. KOLDOVSKY, O., FALTOVA, E., HAHN, P., and VACEK, Z. (1960). “The Functional Development of the Gastrointestinal Tract in Rats in the Development of Homeostasis” (P. Hahn, ed.). Publishing House of the Czechoslovak Academy of Science, Prague. KOLDOVSKY, O., HAHN, P., MELICHAR, V., NOVAK, M., PROCHAZKA, P., ROKOS, J., and VACEK, Z. (1963). Absorption and transport of lipids from the
in Newborn
Rat Intestine
13
small intestine of infant rats. In “Biochemical Problems of Lipids” (A. C. Frazer, ed.), pp. 162171. Elsevier, Amsterdam. LADMAN, A. J., PADYKULA, H. A., and STRAUSS, E. W. (1963). A morphological study of fat transport in normal human jejunum. Amer. J. Anat. 112, 389-420. LEBLOND, C. P., and STEVENS, C. E. (1948). The constant renewal of the intestinal epithelium in the albino rat. Anat. Rec. 100, 357-378. LIPKIN, M. (1973). Proliferation and differentiation of gastrointestinal cells. Physiol. Reu. 53,891-915. MAKELA, O., and NOSSAL, G. (1962). Autoradiographic studies on the immune response. II. DNA synthesis amongst single antibody-producing cells. J. Exp. Med. 115, 231-244. MILLINGTON, P. F., and BROWN, A. C. (1967). Electron microscope studies of the distribution of phosphatase in rat intestinal epithelium from birth to ten days after weaning. Histochemistry 8,109-121. O’CONNOR, T. M. (19661. Cell dynamics in the intestine of the mouse from late fetal life to maturity. Amer. J. Anat. 118, 525-536. OLEDZKA-SLOTWINSKA, H., and DESMET, V. J. (1971). Electron microscopic and cytochemical study on the role of Golgi elements and plasma membrane of enterocytes in the intestinal lipid transport. Histochemistry 28, 276-287. OVERTON, J. (19651. Fine structure of the free cell surface in developing mouse intestinal mucosa. J. Exp. 2001. 159, 195-201. PADYKULA, H. A. (1962). Recent functional interpretations of intestinal morphology. Fed. Proc. 21, 873-879. PALAY, S. L., and KARLIN, L. J. (1959). An electron microscopic study of the intestinal villus II. The pathway of fat absorption. J. Biophys. Biochem. Cytol. 5, 373-384. PEARSE, A. G. E. (1961). “Histochemistry, Theoretical and Applied.” Little, Brown and Co., Boston. PHELPS, P. C., RUBIN, C. E., and LUFT, J. H. (1964). Electron microscopic techniques for studying absorption of fat in man with some observations on pinocytosis. Gastroenterology 46, 134-156. RODEWALD, R. (1970). Selective antibody transport in the proximal small intestine of the neonatal rat. J. Cell Biol. 45, 635-640. RODEWALD, R. (1973). Intestinal transport of antibodies in the newborn rat. J. Cell Biol. 58, 189211. R~STGAARD, J., and BARRNETT, R. J. (1965). Fine structural observations of the absorption of lipid particles in the small intestine of the rat. Anat. Rec. 152, 325-349. SENIOR, J. R. (1964). Intestinal absorption of fats. J. Lipid Res. 5, 495-521. STRAUSS, E. W. (1968). Morphological aspects of triglyceride absorption. In “Handbook of Physiology, Alimentary Canal” (C. F. Code and W. Heidel,
14 eds.), Vol. 3, pp. 1377-1406. Williams and Wilkins, Baltimore. TRIER, J. S., and RUBIN, C. E. (1965). Electron microscopy of the small intestine. A review. Gastro-
enterolopy 49. 574-603. VACEK, ZT, HAHN, P., Histological study of intestine, liver and ministration to rata
and KOLWVSKY, 0. (1962). fat distribution in the small lungs following oral fat adof different postnatal ages.
Czech.
Morphol.
10, 30-45.
WESSELS, N. K. (1964). DNA synthesis, differentiation in pancreatic acinar J. Cell Biol. 20, 415-433.
mitosis, cells in
and
vitro.
WRIGHT, N. A., AL-DEWACHI, H. S., APPLETON, D. R., and WATSON, A. J. (1975). Cell population kinetics in the rat jejunal crypt. Cell Tissue Kinet. 8, 361-368.
57, 1-14
BIOLOGY
(19771
Lipid Absorption MILLIE Department Received
Rat Intestine
I. FERLATTE AND FRANCES J. ZEMAN
of Nutrition, April
in Newborn
8, 1976;
University nrrepted
of California, in revised
Davis, form
California
December
95616
20,1976
There appear to be three distinct regions of cellular maturation within the newborn intestinal epithelium. As crypt cells move onto the lower villus surface, they become transition cells which in turn differentiate into mature villus-absorptive cells. In order to study lipid absorption in cells of each region, proximal segments of intestines of both suckled and corn oilinfused newborn rats were prepared by routine light and electron microscopic methods. Crypt cells were capable of division and, perhaps, of some absorption, whereas villus cells were capable only of absorption. Transition cells were capable of both division and lipid absorption. INTRODUCTION
investigated the morphological and histo chemical characteristics and functional capabilities of transition cells. They appear to be morphologically and functionally distinct from both villus and crypt cells, and they do not seem to correspond to any adult intestinal epithelial cells. This paper describes lipid absorption by intestinal cells from the newborn proximal small intestine and characterizes the function and morphology of transition cells.
Intestinal absorption processes have been shown to differ in neonatal and adult rats (Halliday, 1955; Brambell, 1958). Although there have been many investigations of lipid absorption in adult rat intestine (Palay and Karlin, 1959; Rostgaard and Barrnett, 1965; Cardell et al., 1967; Jersild and Clayton, 1971), and intestinal absorption of various substances has been studied in neonatal and suckling rats (Clark, 1959; Vacek et aZ., 1962; Koldovsky et al., 1963; Graney, 1968; Cornell and Padykula, 1969; Rodewald, 1970, 1973), few have examined lipid absorption in young rats (Vacek et al., 1962; Koldovsky et al., 1963; Cornell and Padykula, 1969). Since the diet of suckling rats is rich in lipid, and since lipid absorption in the mammalian tract occurs primarily within the proximal end of the small intestine (Borgstrom et al., 1957; Johnston, 1959; Dawson and Isselbacher, 1960), we investigated lipid absorption within the proximal intestine of newborn rats. Within the proximal intestinal epithelium of newborn rats, there appears to be a region composed of cells that are in transition from crypt to villus cells. These transition cells are located above the crypts along the lower edges of the villi. We have Copyright All rights
0 1977 by Academic Press, of reproduction in any form
Inc. reserved.
MATERIALS
AND
METHODS
Newborn rats of the Sprague-Dawley strain, weighing 6.4 * 0.3 g were used. Three groups were studied: unsuckled animals, suckled animals, and unsuckled animals infused with corn oil. The proximal small intestines of unsuckled animals were fixed in Bouin’s solution and prepared for light microscopy according to routine procedures. Paraffin sections were cut at 6 pm and stained with hematoxylin and eosin. To differentiate transition cells from crypt-top cells we histochemically analyzed epithelial cells for the presence of alkaline phosphatase, since, in newborn rats, the upper boundaries of the crypts are poorly defined. The proximal intestines of unsuckled animals were frozen in isopentane, chilled in liquid
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ISSN
0012-1606
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DEVELOPMENTAL
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nitrogen, and sectioned at 10 pm. The sections were incubated for 1 hr at room temperature using naphthol AS-MX phosphoric acid as the substrate and red violet LB salt as the chromogen (Pearse, 1961). The sections were counterstained with methyl green and examined the same day. Pieces, 1-2 mm long, were excised from the proximal small intestine of suckled animals about 2 cm from the pyloric sphincter and were fixed in 2.7% glutaraldehyde buffered with 0.13 M sodium cacodylate, pH 7.4, for 1 hr at room temperature. Tissues were postfixed in 1% osmium tetroxide and 0.1 M sodium cacodylate for 1 hr at 4”C, rinsed in buffer, dehydrated through an ethanol series to propylene oxide, and embedded in Epon. Thick sections (1.5 pm) were stained with methylene blue and azure II. Thin sections were stained with uranyl acetate and lead citrate or uranyl acetate alone and were examined with Zeiss 9 and RCA-EMU 3G electron microscopes. Unsuckled newborn animals were anesthetized with sodium pentobarbital, and 0.1 ml of corn oil was infused into the small intestine via a cannula through the stomach and pyloric sphincter. Animals were sacrificed 1 or 3 hr later, and the intestinal tissue was processed as just described. RESULTS
Light
Microscopy
Crypt cells in newborn rats had basophilic cytoplasm and small intensely stained nuclei. A brush border was not evident. Mitoses were frequently observed within the crypts. Cells within the crypts did not reveal a positive response for alkaline phosphatase. On the other hand, intestinal absorptive cells in newborn rats were tall columnar cells with wide brush borders and large rounded nuclei. The apical portions of villus cells showed a positive response for alkaline phosphatase. Between these two types of cells, an intermediate type was observed. We have
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chosen to call these lower villus cells “transition cells”, since they appear to be a distinct cell type that occurs during differentiation in newborn rat intestinal epithelium. These epithelial cells, located along the lower portion of the villus, were tall with elongated nuclei. The cytoplasm was composed of basophilic and acidophilic areas, and the cells had a narrow brush border. These cells gave a positive reaction for alkaline phosphatase on their apical surfaces, distinguishing them from crypt cells. There were numerous instances of mitosis in these cells (Fig. 11, indicating that they were capable of division. After suckling, a few crypt cells were observed to contain osmiophilic droplets. Droplets were rarely visible within mature absorptive cells, and, in the rare instance when droplets were observed, they were generally in cells at the villus tips. Transition cells contained many large osmiophilic droplets distributed throughout the cytoplasm (Fig. 2). Figures 3 and 4 show a transition cell undergoing mitosis in which the cytoplasm contains several droplets. The osmiophilic droplets appeared to be lipid, but since the presence of lipid within cells along the lower villus surface had not been previously described, it was necessary to determine if the transition cells would absorb lipid. One hour after infusion of corn oil, there were no large lipid droplets within crypt or transition cells. However, following a 3-hr exposure to corn oil, we observed many lipid droplets within transition cells similar to those seen in transition cells from suckled animals. Electron
Microscopy
Crypts in newborn rat intestines were not well developed, and their upper limits were not well defined. Crypt cells had sparse stubby microvilli, numerous free riand mitochondria with less bosomes, rough or smooth endoplasmic reticulum than that found in villus cells (Fig. 5).
FERLATTE
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FIG. 1. Light micrograph of villi of unsuckled animal. The top of the crypt is marked with an arrow. A transition cell is undergoing mitosis (M). Hematoxylin and eosin. x480.
After a l-hr exposure to corn oil, there were no large lipid droplets observed within crypt cells, but, after a prolonged exposure to lipid, some crypt cells contained lipid droplets. Cells undergoing mitosis were observed to contain large 1.5 pm-diameter lipid droplets. Pinocytosis of lipid was not observed in any crypt cells, though small lipid droplets, some unbound and others contained within apical cisternae, were observed near the cell surface. Large unbound lipid droplets were formed by coalescence of small lipid droplets. Small lipid droplets were observed within Golgi vacuoles, but saccules remained flattened. Some chylomicra were observed within extracellular spaces, but were far less numerous than were the chylomicra observed within the extracellular space between villus cells.
in Newborn
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3
FIG. 2. Light micrograph of villi of suckled animal. Transition cells contain numerous lipid droplets (L) shown here as dark inclusions. Only a few absorptive cells appear to contain lipid. Cells deep in the crypts (Cl are basophilic and do not contain lipid. Methylene blue and azure II. x 150.
With low power electron microscopy, the brush border of mature absorptive cells was observed to be composed of long narrow microvilli. Although microfilaments extended from the microvilli into the underlying cytoplasm, there were no perpendicular connecting microfilaments to form a well defined terminal web (Fig. 6). The lateral cell membranes were closely apposed and convoluted. Following suckling, the cells contained large numbers of small lipid droplets, predominantly in the supranuclear cytoplasm. Golgi bodies were composed of enlarged saccules containing numerous chylomicra. Occasionally, chylomicra were seen within the supranuclear intercellular space; however, most of the intercellular lipid was below the level of the nuclei. After suckling or after corn oil infusion, there were large lipid droplets in the intestinal lumen. Luminal lipid was surrounded by a light halo covered with an
DEVELOPMENTAL
BIOLOGY
FIG. 3. Light micrograph of suckled animal. Transition cells contain numerous osmiophilic lipid droplets. Crypt cells (0 do not contain lipid droplets. Due to an oblique cut, the intestinal lumen does not extend to the base of the crypt. A transition cell containing lipid is undergoing mitosis (Ml. Methylene blue and azure II. x 375. FIG. 4. Higher power micrograph of the mitotic transition cell (M) in Fig. 3. x 600.
osmiophilic rim. Where the lipid was in contact with microvilli, the halo and rim appeared disrupted, and the lipid outline lost its spherical quality (Fig. 7). The microvilli did not appear to come into direct contact with the lipid droplet; rather, they were connected by fibrils composing the cell coat. Pinocytotic vesicles were few and did not appear to contain lipid. Smooth apical vesicles were observed to contain lipid droplets. Deeper in the cytoplasm, rough endoplasmic reticulum (RER) was often observed to contain lipid droplets (Fig. 8). Mitochondria were numerous and closely surrounded by RER. Some lipid droplets were covered with or surrounded by fine electron-dense parti-
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cles. These particles were due to the lead stain, as they were not present when the lead stain was omitted. Not all lipid droplets reacted with the lead to the same degree; while some droplets were sharply delimited by numerous particles, others had only a few particles overlying them or no depositions. Lipid droplets that were enclosed within endoplasmic reticulum were not observed to have a heavy lead deposition. Unbound droplets were densely surrounded by the particles, and, perhaps, the surrounding membrane inhibited a heavy deposition of lead about the periphery. Golgi bodies had enlarged vacuoles containing chylomicra, still delimited by lead particles (Fig. 9). After chylomicra were transported to the extracellular space, they did not accumulate the electron-dense particles about their peripheries: The luminal surfaces of transition cells were composed of numerous short microvilli, intermediate in length between those of villus and crypt cells and covered with a fuzzy layer. Lateral cell membranes were closely apposed and connected at the apical end by junctional complexes. Adjacent cells exhibited some lateral interdigitations, whereas some of the lateral cell membranes appeared straight. The terminal web was not well formed. Smooth vesicles were present in the apical portion of the cell, as well as dense bodies and multivesicular bodies. Mitochondria and numerous free ribosomes were seen throughout the cell, but free ribosomes were less numerous than in crypt cells. RER, generally in close conjunction with mitochondria, was more abundant than in crypt cells. Electron microscopy confirmed that, after 1 hr of exposure to corn oil, there was no appreciable amount of absorbed lipid within transition cells, but, after a 3-hr exposure to the oil, transition cells were filled with numerous lipid droplets of various sizes (Fig. 10). Some of the droplets were membrane-bound and others were free in the cytoplasm. Large lipid droplets
FERLATTE
AND ZEMAN
FIG. 5. Crypt cells from suckled newborn Golgi apparatus (G) is composed of flattened present. x 17,760.
Absorption
in Newborn
Rat Intestine
contain numerous mitochondria saccules and small vacuoles.
were formed by coalescing smaller ones. Cellular organelles were displaced by enormous lipid droplets (Figs. 10 and 111. A few small droplets were observed within the cell adjacent to the apical cell surface. Although pinocytotic vesicles were observed, none appeared to contain lipid. Lipid droplets were often in close conjunction with mitochondria and RER. Some of the droplets were contained within the RER. The Golgi apparatus was active, as indicated by enlarged vacuoles, but not to the same extent as in mature absorptive cells. Extracellular lipid which had been transported through the cell was present as a few discrete droplets or as droplets so numerous that the entire extracellular space was occluded.
and free ribosomes A few lipid droplets
5
(RI. The (L) are
DISCUSSION
Currently, two methods of lipid absorption in the small intestine have been proposed, passive diffusion of hydrolyzed lipid and bulk pinocytosis of lipid. Briefly, passive diffusion of lipid occurs as follows. Intestinal luminal content consists of an oily phase and a micellar phase (Hofmann and Borgstrom, 19621. Components of the oily phase, triglycerides, are transformed into a micellar solution of monoglycerides and free fatty acids by the action of pancreatic lipase and bile salts. Monoglycerides and free fatty acids then passively diffuse through the plasma membrane (Johnston and Borgstrom, 1964; Jersild, 1966; Cardell et al., 1967; Johnston, 19681, where they are resynthesized into triglyc-
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DEVELOPMENTAL
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FIG. 6. Low power electron micrograph of villus-absorptive extend into the cytoplasm, but a well-formed terminal web bound lipid droplets (L) are present. x 17,760.
erides within the SER (Cardell et al ., 1967; Strauss, 1968). Although pinocytosis of lipid has been reported by some investigators (Palay and Karlin, 1959; Ashworth et al., 1960; Cornell and Padykula, 1969), others have not observed pinocytosis of lipid following ingestion of fat (Ladman et. al., 1963; Phelps et al., 1964; Cardell et al., 1967; Dermer, 1967; Oledzka-Slotwinska and
57, 1977
cells of a suckled animal. Microfilaments is not evident. Numerous small membrane-
Desmet, 1971). Consequently, pinocytosis has not been universally accepted as a major route of lipid absorption (Strauss, 1968). Senior (1964) has pointed out that the two methods of fat absorption, diffusion of micellar components with intracellular resynthesis and bulk pinocytosis of unhydrolyzed lipid, are not mutually exclusive. He suggested that, as large lipid droplets are
FERLATTE
AND ZEMAN
Absorption
hydrolyzed, smaller unhydrolyzed droplets are formed which may be engulfed in apical pits. However, we did not observe small lipid droplets near large luminal lipid droplets, and it has been shown that the apical pits are not involved in pinocytosis of lipid (Cardell et al., 1967; Rodewald, 1970). Since lipid is absorbed more rapidly in lo-day-old rats than in adults, it has been
in Newborn
Rat Intestine
7
proposed that fat absorption in suckling rats differs from that in adult animals (Koldovsky, 1969). It has been suggested that neonatal rats are able to absorb triglycerides intact without prior hydrolysis (Koldovsky et al., 1960). Cornell and Padykula (1969) observed pinocytosis of lipid within ileal cells of neonatal rats. In the newborn rats in this study, we did not observe pinocytosis of lipid nor were we
FIG. 7. A lipid droplet within the intestinal lumen is surrounded by a light halo (H) and an osmiophilic rim CR). Where it is in contact with the glycocalyx, the spherical outline breaks down (arrows). Small lipid droplets CL) are contained within smooth vesicles and unbound lipid KJL) is free in the cytoplasm of a villus absorptive cell. x 14,400.
8
DEVELOPMENTAL
FIG. 8. Columnar cell of suckled and mitochondria (M). Lipid droplets dense particles. x 32,600.
BIOLOGY
animal showing (L) are within
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close apposition of rough endoplasmic reticulum (RER) the RER. Unbound lipid (UL) is surrounded by electron-
able to identify lipid within the terminal web area; we did so only deeper in the cytoplasm, suggesting that newborn duodenal cells absorb lipid by diffusion across the plasma membrane. Crypt cells, distinguishable from absorptive cells because of cytoplasmic basophilia and from villus cells by the presence of alkaline phosphatase (Padykula, 1962; Overton, 1965; Millington and Brown, 1967; Oledzka-Slotwinska and Desmet, 19711, were similar to those described by Trier and Rubin (1965). The cytoplasmic basophilia of crypt cells is due to a high concentration of RNA (Padykula, 1962) existing as free ribosomes, a characteristic of undifferentiated cells. After a long exposure to fat, only a few crypt ceils contained lipid, resulting possibly from absorption or from synthesis of lipid from simple precursors derived in part from the plasma. Build-up of lipid within immature intestinal cells has been attributed to storage for intracellular use (Dobbins, 1969). In the case of crypt cells, glycerolipid is necessary for the synthesis of membrane phospholipid for newly formed cells (Gang1
and Ockner, 1974). The ultrastructure of absorptive cells from the proximal small intestine of newborn rat pups appeared similar to that of previous descriptions of mature villus cells (Palay and Karlin, 1959; Dunn, 1967; Cardell et al., 1967; Strauss, 1968; Rodewald, 1973). Since the ultrastructure of lipid absorption has been described in detail (Cardell et al., 1967; Strauss, 1968; Cornell and Padykula, 19691, only those observations that add new information will be discussed here. The discussion will follow the path of lipid as it moves from the intestinal lumen through the cell to enter the extracellular space. Luminal lipid did not come into direct contact with the apical cell membrane, but was separated by the glycocalyx covering the surface of the microvilli. The glycocalyx may play a role in lipid absorption, as evidenced by the intimate relationship between the glycocalyx and lipid droplets. The outlines of luminal lipid droplets were round and smooth except where they were in contact with the glycocalyx, suggesting
FERLATTE
AND ZEMAN
Absorption
FIG. 9. An electron micrograph of a Golgi body (G) within an absorptive cell. The chylomicra within the Golgi are delimited by electron-dense particles. The chylomicra (CH) within the extracellular space (ES) do not aggregate lead particles. x 33,300.
that the glycocalyx may assist in the breakdown of lipid. The close relationship between RER and mitochondria has been previously reported (Cardell et al., 1967; Hayward, 1967; Friedman and Cardell, 1972). Smooth and rough ER have been shown to be in continuity in the adult animal, and lipid droplets have occasionally been observed within RER, but RER has not been considered a path-
in Newborn
Rat Intestine
9
way of lipid from SER (Cardell et al., 1967; Dobbins, 1969; Friedman and Cardell, 1972). Cardell et al. (1967) attributed the presence of lipid droplets within RER to its synthesis in adjacent SER. We observed numerous instances of lipid droplets within RER and feel that RER plays a role in the transport of lipid through the cell, perhaps in transporting the lipid from the SER to the Golgi body. Rough ER contributes the protein component of chylomicra (Dobbins, 19691, and it has been suggested previously (Senior, 1964) that the lipid droplet moves through the RER to pick up its protein coating. Electron-dense particles surrounding and covering the lipid droplet were apparently artifacts of lead staining as previously demonstrated (Rostgaard and Barrnett, 1965). However, it appeared that adjacent lipid droplets within the cell cytoplasm exhibited different affinities to the lead stain, perhaps due to differences in surface properties of the droplets. The differences in surface properties may be due to the presence of two types of intracellular lipid droplets, those bound within endoplasmic reticulum and unbound or matrix lipid (Friedman and Cardell, 1972). ERbound lipid did not appear to accumulate heavy lead deposits, whereas unbound lipid droplets were heavily stained by the lead. Although we could not discern a membrane about those droplets with a heavy deposition of lead, there exists the possibility that the lead deposition may have prevented our viewing it. The chylomicra within the Golgi body were outlined with dense particles, but, once the chylomicra had been transported from the cell, they were not. A carbohydrate moiety is added to chylomicra within the Golgi body (Friedman and Cardell, 1972), and this may alter their surface properties so the lead would not precipitate about their peripheries. The morphological appearance of lipid absorption within transition cells was unlike that within either crypt or absorptive
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FIG. 10. A low power electron micrograph of transition cells from a suckled newborn. There are numerous lipid droplets (LI of various sizes throughout the cytoplasm and the extracellular space is filled with chylomicra (CH). x 6600.
FERLATTE
AND ZEMAN
Absorption
in Newborn
FIG. 11. Transition cells from suckled newborn animal with large lipid cytoplasm. The Golgi body (G), though small, contains chylomicra (arrow). ous than in crypt cells. x 11,571.
cells. Only in transition cells were there massive accumulations of lipid within the cells. It is possible that the transition cells were not absorbing lipid from the intestinal lumen, but rather were synthesizing it from simple precursors derived from the plasma as crypt cells do (Gang1 and Ockner, 1974). However, as transition cells will soon mature into absorptive cells, it
11
Rat Intestine
droplets within Free ribosomes
the supranuclear are less numer-
seems reasonable that the cells have the capability to absorb and transport lipid through the cells. However, transition cells appeared restricted in their capacity to transport lipid through the cell. Rough ER, the site of protein synthesis, was less abundant in transition cells than in absorptive cells, and the Golgi apparatus was less active as judged by fewer cristae and
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DEVELOPMENTAL
BIOLOGY
smaller vacuoles. Both of these organelles have been shown to play important roles in the formation of chylomicra (Isselbather, 1965; Dobbins, 1969). Friedman and Cardell (1972) utilizing puromycin to block protein synthesis, produced absorptive cells filled with lipid, similar to transition cells of suckled rats. They proposed that the action of puromycin prevented the RER from synthesizing membranes destined for the Golgi apparatus, causing a reduction of Golgi membranes which normally envelop the chylomicra, and suggested that, without the enveloping membranes, the cells were unable to release the chylomicra, thus causing a buildup within the cells. Differentiation of transition cells has progressed to allow for some chylomiera formation, as evidenced by the large numbers of chylomicra within the extracellular space at the bases of the cells, but the cells have not acquired the mature complements of RER and Golgi apparatus that are present in absorptive cells. Progressive differentiation of epithelial cells occurs as they migrate from the crypt along the villus (Padykula, 1962; Lipkin, 1973; Cheng and Leblond, 1974). Transition cells in the newborn intestine appear to represent an intermediate cell type in the maturation process from crypt to villus. Transition cells, unlike crypt and villus cells, are capable of both absorption and division. The presence of mitotic figures within the intestinal epithelium has been described as occurring only within the confines of crypts (Leblond and Stevens, 1948; Cairnie et al., 1965; O’Connor, 1966; Wright et al., 1975). There does not seem to be an overlap of functional capabilities in adult intestinal epithelium, as crypt cell proliferative capability ceases prior to attainment of villus absorptive function. In most organ systems proliferation and differentiation are mutually exclusive, but, in some instances, a degree of overlap can be seen. In developing pancreas, mitotic cells contain zymogen (Wessells, 19641, and plasma cells can develop
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antibody before the cessation of cell proliferation (Makela and Nossal, 1962). Unlike adult intestinal cells which lose crypt mitotic function prior to gaining villus absorptive and transport functions, it appears that newborn intestinal cells retain their ability to divide while their capability to absorb and transport nutrients increases. This situation is advantageous for newborn animals with their shorter villi and shallow ‘crypts, as they are able to utilize all of their villus cells plus transition cells to some extent for absorption, and they have a proliferating pool of cells composed of both crypt and transition cells. This research was supported, in part, by U.S. Public Health Service Grant No. HD-06465 from the National Institute of Child Health and Human Development. REFERENCES C. T., STEMBRIDGE, V. A., and SANDERS, E. (1960). Lipid absorption, transport and hepatic assimilation studied with electron microscopy. Amer. J. Physiol. 198, 1326-1328. BORGSTR~M, B., DAHLQUIST, A., LUNDH, G., and SJOVALL, J. (1957). Studies of intestinal digestion and absorption in the human. J. Clin. Invest. 36, X21-1536. BRAMBELL, F. W. R. (1958). The passive immunity of the young mammal. Biol. Reu. 33, 488-531. CAIRNIE, A. B., LAMERTON, L. F., and STEEL, G. G. (1965). Cell proliferation studies in the intestinal epithelium ofthe rat. Exp. Cell Res. 39,528-538. CARDELL, R. R., JR., BADENHAUSEN, S., and PORTER, K. R. (1967). Intestinal triglyceride absorption in the rat. An electron microscopical study. J. Cell Biol. 34, 123-155. CHENG, H., and LEBLOND, C. P. (1974). Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. I. Columnar cell. Amer. J. Anat. 141, 461-479. CLARK, S. L., JR. (1959). The ingestion of proteins and colloidal materials by columnar absorptive cells of the small intestine in suckling rats and mice. J. Biophys. Biochem. Cytol. 5, 41-49. CORNELL, R., and PADYKULA, H. A. (1969). A cytological study of intestinal absorption in the suckling rat. Amer. J. Anat. 125, 291-316. DAWSON, A. M., and ISSELBACHER, K. J. (1960). The esterification of palmitate-l-‘4C by homogenates of intestinal mucosa. J. Clin. Inuest. 39, 150-160. DERMER, G. B. (1967). Ultrastructural changes in the microvillous plasma membrane during lipid
ASHWORTH,
FERLATTE
AND ZEMAN
A bsoi rption
absorption and the form of absorbed lipid: An in viva study. J. Ultrastruct. Res. 20, 51-71. DOBBINS, W. 0. III (1969). Morphologic aspects of lipid absorption. Amer. J. Clin. Nutr. 22,257-265. DUNN, J. S. (1967). The fine structure of the absorptive epithelial cells of the developing small intestine of the rat. J. Anat. (London) 101, 57-68. FRIEDMAN, H. I., and CARDELL, R. R., JR. (1972). Effects of puromycin on the structure of rat intestinal epithelial cells during fat absorption. J. Cell Biol. 52, 15-40. GANGL, A., and OCKNER, R. (1974). Intestinal metabolism of plasma free fatty acid. Clin. Res. 22, 358A. GRANEY, D. 0. (1968). The uptake of ferritin by ileal absorptive cells in suckling rats. An electron microscope study. Amer. J. Anat. 123, 227-254. HALLIDAY, R. (1955). The absorption of antibodies from immune sera by the gut of the young rat. Proc. Roy. Sot. B 143, 408-413. HAYWARD, A. F. (1967). Changes in fine structure of developing intestinal epithelium associated with pinocytosis. J. Anat. 102, 57-70. HOFMANN, A. F., and BORGSTRBM, B. (1962). Physice-chemical state of lipids in intestinal content during their digestion and absorption. Fed. Proc. 21, 43-50. ISSELBACHER, K. J. (1965). Metabolism and transport of lipid by intestinal mucosa. Fed. Proc. 24, 16-22. JERSILD, R. A., JR. (1966). A time sequence of fat absorption in the rat jejunum. Amer. J. Anat. 118, 135-162. JERSILD, R. A., JR., and CLAYTON, R. T. (1971). A comparison of the morphology of lipid absorption in the jejunum and ileum of the adult rat. Amer. J. Anat. 131, 481-503. JOHNSTON, J. M. (1959). Site offatty acid absorption. Proc. Sot. Exp. Biol. Med. 100, 669-670. JOHNSTON, J. M. (1968). Mechanism of fat absorption. In “Handbook of Physiology, Alimentary Canal” (C. F. Code and W. Heidel, eds.), Vol. 3, pp. 1353-1375. Williams and Wilkins, Baltimore. JOHNSTON, J. M., and BORGSTR~M, B. (1964). The intestinal absorption and metabolism of micellar solutions of lipids. Biochem. Biophys. Acta 84, 412-423. KOLDOVSKY, 0. (1969). “Development of the Functions of the Small Intestine in Mammals and Man,” p. 85. Karger, Basel. KOLDOVSKY, O., FALTOVA, E., HAHN, P., and VACEK, Z. (1960). “The Functional Development of the Gastrointestinal Tract in Rats in the Development of Homeostasis” (P. Hahn, ed.). Publishing House of the Czechoslovak Academy of Science, Prague. KOLDOVSKY, O., HAHN, P., MELICHAR, V., NOVAK, M., PROCHAZKA, P., ROKOS, J., and VACEK, Z. (1963). Absorption and transport of lipids from the
in Newborn
Rat Intestine
13
small intestine of infant rats. In “Biochemical Problems of Lipids” (A. C. Frazer, ed.), pp. 162171. Elsevier, Amsterdam. LADMAN, A. J., PADYKULA, H. A., and STRAUSS, E. W. (1963). A morphological study of fat transport in normal human jejunum. Amer. J. Anat. 112, 389-420. LEBLOND, C. P., and STEVENS, C. E. (1948). The constant renewal of the intestinal epithelium in the albino rat. Anat. Rec. 100, 357-378. LIPKIN, M. (1973). Proliferation and differentiation of gastrointestinal cells. Physiol. Reu. 53,891-915. MAKELA, O., and NOSSAL, G. (1962). Autoradiographic studies on the immune response. II. DNA synthesis amongst single antibody-producing cells. J. Exp. Med. 115, 231-244. MILLINGTON, P. F., and BROWN, A. C. (1967). Electron microscope studies of the distribution of phosphatase in rat intestinal epithelium from birth to ten days after weaning. Histochemistry 8,109-121. O’CONNOR, T. M. (19661. Cell dynamics in the intestine of the mouse from late fetal life to maturity. Amer. J. Anat. 118, 525-536. OLEDZKA-SLOTWINSKA, H., and DESMET, V. J. (1971). Electron microscopic and cytochemical study on the role of Golgi elements and plasma membrane of enterocytes in the intestinal lipid transport. Histochemistry 28, 276-287. OVERTON, J. (19651. Fine structure of the free cell surface in developing mouse intestinal mucosa. J. Exp. 2001. 159, 195-201. PADYKULA, H. A. (1962). Recent functional interpretations of intestinal morphology. Fed. Proc. 21, 873-879. PALAY, S. L., and KARLIN, L. J. (1959). An electron microscopic study of the intestinal villus II. The pathway of fat absorption. J. Biophys. Biochem. Cytol. 5, 373-384. PEARSE, A. G. E. (1961). “Histochemistry, Theoretical and Applied.” Little, Brown and Co., Boston. PHELPS, P. C., RUBIN, C. E., and LUFT, J. H. (1964). Electron microscopic techniques for studying absorption of fat in man with some observations on pinocytosis. Gastroenterology 46, 134-156. RODEWALD, R. (1970). Selective antibody transport in the proximal small intestine of the neonatal rat. J. Cell Biol. 45, 635-640. RODEWALD, R. (1973). Intestinal transport of antibodies in the newborn rat. J. Cell Biol. 58, 189211. R~STGAARD, J., and BARRNETT, R. J. (1965). Fine structural observations of the absorption of lipid particles in the small intestine of the rat. Anat. Rec. 152, 325-349. SENIOR, J. R. (1964). Intestinal absorption of fats. J. Lipid Res. 5, 495-521. STRAUSS, E. W. (1968). Morphological aspects of triglyceride absorption. In “Handbook of Physiology, Alimentary Canal” (C. F. Code and W. Heidel,
14 eds.), Vol. 3, pp. 1377-1406. Williams and Wilkins, Baltimore. TRIER, J. S., and RUBIN, C. E. (1965). Electron microscopy of the small intestine. A review. Gastro-
enterolopy 49. 574-603. VACEK, ZT, HAHN, P., Histological study of intestine, liver and ministration to rata
and KOLWVSKY, 0. (1962). fat distribution in the small lungs following oral fat adof different postnatal ages.
Czech.
Morphol.
10, 30-45.
WESSELS, N. K. (1964). DNA synthesis, differentiation in pancreatic acinar J. Cell Biol. 20, 415-433.
mitosis, cells in
and
vitro.
WRIGHT, N. A., AL-DEWACHI, H. S., APPLETON, D. R., and WATSON, A. J. (1975). Cell population kinetics in the rat jejunal crypt. Cell Tissue Kinet. 8, 361-368.
