JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 16156-68 (1990)
Fasting Induces Modifications of the Endoplasmic Reticulum in Intestinal Cells JENNIFER McLEESE AND MICHEL BERGERON Department of Physiology, UniuersitQde Montrkal, Montrkal H3C 357, QuQbec,Canada
KEY WORDS
Epithelial cells, Transport, Intestinal cell, Flounder
ABSTRACT
The polymorphism of the endoplasmic reticulum (ER) in epithelial cells with different transport functions such as the enterocyte suggests that the ER may be involved in some way in molecular transport. To further access this possibility, we examined the ER from the intestine of winter flounder, Pseudopleuronectes americanus, a species which undergoes an annual fast of approximately 6 months’ duration, a time during which previous work indicates nutrient carrier number does not change. Fish from June (feeding) and January (8-10 weeks fasted) were sampled. Tissues from the pyloric caeca, foregut, midgut, and hindgut were prepared for electron microscopy using two techniques of staining. Cell height was unaltered in any section, although microvillar length shortened variably. Cellular organization, including position of nuclei, number and distribution of mitochondria, and presence of basolateral membranes, did not change. The ER appeared equally abundant in June and January. However, use of the osmium impregnation technique, which is specific for ER cisternal contents, revealed a change in the impregnation of ER, from a heavily impregnated network in summer t o little or no impregnation in winter. These results suggest that a shift in function of the ER had occurred when nutrient transport ceased, and supports arole of the ER in nutrient transport.
INTRODUCTION
metal impregnation technique (Bergeron and Thiery, 1981; Bergeron et al., 1978; Mollgard and Rostgaard, The extensive organization of the ER in transport 1978; Thiery et al., 1983) as well as other histochemical epithelial cells and its polymorphic characteristics techniques (Novikoff et al., 19831, offers a logical morhave been underestimated if not ignored. In any model phological basis for the integrated functions that the of transport epithelial cell, the plasma membranes, RER, SER, and Golgi apparatus appear to carry out in junctional complexes, and mitochondria are always absorptive cells (see for review Madara and Trier, portrayed, whereas the ER is seldom, if ever, repre- 1987). sented. Yet, in most epithelial cells the ER is a wellThe ER is portrayed, almost exclusively, as the site organized network (Bergeron and Thiery, 1981; Berg- of protein synthesis and its schematic representation eron et al., 1978, 1987, 1988; Berthelet et al., 1987; follows Palade’s concepts based on the pancreatic aciMollgard and Rostgaard, 1978; Novikoff et al., 1983; nar cells, a large producer of proteins. However, all Palade, 1955; Porter, 1953; Porter and Palade, 1957; eukaryotic cells have an ER and a Golgi apparatus, yet Thiery 1979; Thiery et al., 1983). In both the rat jeju- protein secretion is not their primary function. Renum and proximal nephron, the ER organization is al- cently the ER was found to be a reservoir for calcium most identical: a continuous network of canaliculi and ions which are liberated following hormonal or pharfenestrated saccules extending from the apex, below macological stimuli which initiate the phosphoinositol the microvilli, to the lateral and basilar plasma mem- cascade a t the V, receptors (Bayerdorffer et al., 1984; branes and sending many projections to the nuclear Berridge, 1984; Muallem et al., 1985). The absence of envelope. Circular, or at times flattened, canaliculi of both V1 receptors (Jard, 1981) and of a well-organized irregular diameters varying between 0.005-0.03 FM, ER (Bergeron et al., 1987) in principal cells of the renal run more-or-less parallel to the long axis of the cell. collecting tubules is an intriguing finding since calFenestrated saccules, possessing fairly regular perfo- cium liberation from the ER is crucial in the inositol rations, averaging 0.03 FM, are interposed on the path pathway. of the canaliculi; these fenestrated saccules surround In epithelial cells, the ER is also a key element in the apical vacuoles and mitochondria as well as cytoplas- regulation of transport carriers and their insertion into mic bodies and vary accordingly in size. The nuclear envelope appears as a specialized part of the ER, which also seems, in thick sections, to be connected to the Golgi apparatus. Along the lateral plasma membranes extensive fenestrated saccules are observed and were Received July 31, 1989; accepted in revised form August 30, 1989 in fact previously described in ultrathin sections as the Address reprint requests to Michel Bergeron, M.D., Departement de physiolparamembranous cisternal system (Ericsson, 1964; ogie, Universit6 de MontrBal, C.P. 6128, Succursale A, Montreal H3C 357, Qu& Erkocak et al., 1963).This ER continuity, shown by the bec. Canada.
0 1990 WILEY-LISS, INC
FASTING-INDUCED MODIFICATION OF ER
basolateral or apical membranes. The fact that the ER appears, in three-dimensional photographs, to constitute a transcytoplasmic route led some investigators (Mollgard and Rostgaard, 1978) to suggest that it may constitute an additional transport system for sodium, a highly questionable hypothesis. Moreover, the ER is not a purely static cellular compartment but shows extensive variations in its osmium staining properties. For example, the evolution of the ER during cellular ascent from the intestinal crypt to the top of the villus is towards a greater complexity (Thiery et al., 1983). At the base of the crypt of Lieberkun, no definite organization is noted; and, most often, only the nuclear envelope and a few canalicular elements are seen. In the mid-crypt, the ER becomes organized, and fenestrated saccules appear and in mature columnar cells the ER finally forms, a transcytoplasmic continuous network of canaliculi and fenestrated saccules. Such an evolution of the ER organization is also noted during renal ontogeny (Gaffiero et al., 1983). The ER impregnation was found to correlate with their physiological status. It can be modified by hormonal factors in prostatic secretory cells (BeaudryLonergan et al., 19851,by chronic metabolic acidosis, or treatment with triamcinolone in proximal tubule cells of the rat kidney (Berthelet et al., 1985). These data suggested that ER impregnation is related to cellular activity and that this technique may provide a test for cell function and organelle interrelations. In jejunal cells, the question arises as to whether ER impregnation is somehow related t o the absorption of nutrients; could the ER function as a transcellular route for movement of molecules such as peptides, amino acids, or glucose (Bergeron and Thiery, 1981; Bergeron et al., 1978; Nordlie, 1979) or sodium (Mollgard and Rostgaard, 1978). In jejunal villi of well-fed rats, cells are generally well-impregnated, but it is not uncommon to see cells with a non-impregnated ER, a phenomenon which appears to be amplified in fasting rats. Because of ethical considerations it is difficult to obtain a definite answer in fasting mammals, and we have therefore examined the ER from the intestine of winter flounder, Pseudopleuronectes americanus, a species which undergoes a natural annual fast of approximately 6 months' duration, a time during which previous work indicates nutrient carrier number per unit surface area does not change (McLeese, 1986). Our results revealed a change in the osmium impregnation of ER, from a heavily impregnated network in summer (feeding) to little or no impregnation in winter (fasting). These results in fishes as well as fragmentary results in the rat suggest that a shift in function of the ER has occurred when nutrient transport ceased, and indirectly support a role of the ER in nutrient transport. MATERIALS AND METHODS Winter flounders were caught from Passamaquoddy Bay (New Brunswick, Canada) by otter trawl and brought to the Huntsman marine laboratory where they were maintained in tanks of running seawater. Fish sampled in the summer were used within 1day of
57
capture; fish for winter sampling were brought to the laboratory in October and held until early January. They were not offered food through this holding period, which corresponds to a natural fasting time (Fletcher et al., 1981; Kennedy and Steele, 1971). Fish were sacrificed by cervical dislocation. The intestines were rapidly excised and segments from the pyloric caeca, foregut, midgut, and hindgut were removed and placed in fixative. Tissues were subsequently prepared by one of two methods: the osmium impregnation technique of Thiery (Thiery, 1979; Thibry et al., 1983) or the lead and copper citrate technique of Thiery and Rambourg (1976).Briefly, osmium impregnation, which stains the cisternal contents of the endoplasmic reticulum, involves fixation in 2.5% glutaraldehyde for 20 min, followed by postfixation in 1%osmium tetroxide for 1.5 hr a t 4°C. Tissue blocks were then left in 1%osmium tetroxide a t 37°C for 3 days. The osmium solution was replaced daily. The lead and copper citrate technique requires fixation in 2.5% glutaraldehyde. Leucine aminopeptidase activity was assessed a t 8°C by the method of Appel(1974). Protein was determined by the method of Lowry et al. (1951). Phenylalanine uptake by emusculated sheets of intestinal mucosa was measured with '*C-radiolabelled phenylalanine using a system based on that described by Lukie et al. (1974). Incubation times was 2 min, followed by a 20 sec rinse in unlabelled flounder Ringer's. Tissue was cut free of the mounting chamber, dried to determine dry weight, then solubilized and counted for radioactivity by liquid scintillation counting. Tritiated mannitol was employed as the unabsorbed marker.
RESULTS Preliminary data have shown that the ER osmium impregnation of jejunal cells was modified by fasting in rats. Figure 1 shows the intensive impregnation of the ER and its rich organization as a network, which was previously described (Thiery et al., 1983). Four days after food was removed, which corresponds to a fasting of about 24 hours (or more), almost all jejunum cells in the mid-crypt still have a well-impregnated ER (Fig. l), while most cells located at the top of the villous are not impregnated with osmium (Fig. 2). Such a striking difference from the well-fed rats may imply that ER has a role in nutrient transport. Because of ethical considerations, we have looked for a species which undergoes a natural fast. We have therefore examined the ER of gut cells of winter flounders, Pseudopleuronectus americanus. Figure 3 is a schematic representation illustrating the various functional and anatomical divisions of the flounder gut, which is much less specialized than the mammalian gut. The gut was arbitrarily divided into four sections, but these were found to be functionally different (McLeese, 1986). Enterocytes of the flounder were tall and narrow. Mitochondria and endoplasmic reticulum (ER) were abundant in the cell apex, but were also present basally (Figs. 4A, 5A). A prominent Golgi apparatus was usually supranuclear. The nucleus was located toward the basal half of the cell, and the nuclear envelope was
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Fig. 1. Mid-crypt of the rat jejunum. Note the extensive ER organization revealed by osmium impregnation after 5 days. Under the brush border, one sees a continuous network of canaliculi and fenestrated saccules which are themselves conected to other canaliculi. Fenestrated saccules are surrounding the mitochondria. The perinuclear membrane is well stained and the pores can be easily seen in
some nuclei (A). Canaliculi are in contact with the nuclear membrane. Note that, in this fasting animal (4 days), all cells of the mid villous are impregnated with osmium. Fasting seems to induce intracellular dilatation. (For further description of the ER of the mammalian jejunum enterocyte, see Thi6ry et al., 1983.) x 2,000.
observed at times to be continuous with the elements of the ER. Microvilli were present at the luminal face of the cells, and intermicrovillous invaginations were observed in all segments of the intestine. Numerous small vacuoles were present immediately beneath the microvilli and in the apical cytoplasm. Microvesicular bodies were common apically in all segments. Lamellar structures, which resembled the lateral plasma membranes in their staining properties, were observed in all segments of the intestine. They are analogous to the basilar infolding seen in many transport epithelia. They were especially abundant basally, but were also present apically in many cells. In many cases, ER cisternae were in close association with mitochondria (Fig. 6). After 8-10 weeks of fasting, the enterocytes did not appear to be shortened, although microvilli were variably shortened and sometimes reduced in number (Figs. 4B,5B). The alterations in the microvillar morphology did not appear to be consistent: in a given region of the gut, adjacent cells showed short and long microvilli. Mitochondria1 profiles were less numerous apically. Microvesicular bodies and intermicrovillous invaginations were still seen in many cells, particularly those in the hindgut. Dense bodies were present in the apical cytoplasm in all segments. Such struc-
tures were not observed in the enterocytes of feeding fish. The lamellar structures of the basilar plasma membranes appeared unchanged by feeding condition. Apical ER appeared to have become more dilated with fasting (Fig. 5B). Large, irregularly shaped cisternae were prominent in many cells, particularly those of the caeca and the foregut (Fig. 6).
Osmium impregnation (Figs. 7-10) Osmium impregnation specifically stains the cisternal contents of the ER. Cut in thick sections, osmium impregnated tissues give an idea of the three dimensional structure of the ER. Fenestrated saccules such as are observed in mammalian intestine (Thiery et al., 1983) were not present in any segment (Figs. 7-10). Impregnation of ER tubules, not associated with the nucleus, differed markedly according to feeding status, and the tubules displayed a high degree of regional polymorphism (Figs. 7-10). The apical ER of the pyloric caeca from feeding flounder could be discerned as large, discrete tubules heavily impregnated with osmium (Fig. 8A). Large, heavily impregnated regions, almost saccules, as well as smaller, fine networks of tubules were observed in the foregut (Fig. 8A). In the midgut, fine, complex networks were observed (Fig. 9A), whereas only large sac-
FASTING-INDUCED MODIFICATION OF ER
59
Fig. 2. Top of the villous of the rat jejunum. Fasting for 4 days. Note that most cells are poorly or not impregnated. One cell shows the extensive normal ER network seen in normal fed animals. Most Golgi are well-impregnated (arrows). Osmium impregnation, x 2,000.
cosal protein concentrations were significantly lower in fasting fish than in feeding fish only in the hindgut.
DIVISIONS OF THE FLOUNDER INTESTINE STOMACH
WLORIC SPHINCTER
Fig. 3.
FOREGUT
PYLORIC CAECA
MIDGUT
HINOGUT
VALVE
Schematic representation of the flounder gut.
cules of osmium deposits were observed in the hindgut (Fig. lOA), without smaller tubules. In fact, some cells had a well-developed ER similar to the rat jejunum. The more basal regions were similar regardless of region, with large deposits of osmium rather than tubular networks. Osmium deposition was much reduced in cells from fasting fish. Fine networks were never observed. However, large deposits occurred in all segments except the hindgut. Osmium impregnation was least reduced by fasting in the foregut.
Physiological characteristics Neither the maximal uptake rate (Jmax) of phenylalanine nor the activity of leucine aminopeptidase decreased in any segment following fasting (Fig. ll).Mu-
DISCUSSION The intestinal mucosa of animals is well known to atrophy disproportionately to the body during starvation. This is thought to be a result of the loss of direct stimulation by nutrients. However, the intestine of a marine fish serves a dual function, acting as an important organ of osmoregulation as well as of digestion. In the winter flounder, the annual winter fast results in a change of function from digestion and osmoregulation to osmoregulation alone (Nonnotte et al., 1986; Skadhauge and Lotan, 1974; Sleet and Weber, 1982). Ion and water transport for osmoregulation has been suggested to ameliorate the effects of fasting on the surface area of the intestine in this fish, and help in the maintenance of the proximodistal gradient of surface area (McLeese and Moon, 1989). The results from the leucine aminopeptidase assays and the phenylalanine uptake experiments suggest that no decrease in enzyme or carrier number per surface area occurred with fasting. Because turnover times for these membrane components are not known, it is difficult to assess whether protein synthesis continued during fasting. However, since protein concentration decreased only in the hindgut, and it was only in the hindgut that osmium impregnation was totally
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Fig. 4. Hindgut of the flounder. Staining with lead and copper citrate and uranyl acetate. A: Feeding. x 20,000. B: Fasting. X 8,500.The ER was again more dilated in B than in A (arrows). Note the microvilli which are smaller in fasting animals than in well-fed flounders.
FASTING-INDUCED MODIFICATION OF ER
Fig. 5. Pyloric caecum of the flounder. Staining with lead and copper citrate and uranyl acetate. A: Feeding. x 13,000. B: Fasting. x 13,000. The ER network is extensive and cisternae are dilated after fasting (arrows). Some cisternae have the aspect of big vacuoles.
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Fig. 6. Foregut of feeding fish. Staining as in Figures 4 and 5 . Photograph showing plasma membranes and extensive ER dilatations. Note the close association between mitochondria and ER, which was described in rat jejunum and proximal nephron. (See Thiery et al., 1983, for further description of organelle relationship.) x 28,000.
abolished, it seems likely that the ER was still active to some extent in the other three intestinal sections. Beaudry-Lonergan et al. (1985) demonstrated a change in the structure and chemistry (indicated by the altered osmium black deposition) of the ER with altered function of the rat prostatic secretory cell. The present study suggests that analogous changes occurred in the enterocytes of the winter flounder intestine. As seen from Figure 4B, the ER of the upper intestine became more dilated during fasting. The appearance of large deposits of osmium black in the caeca, foregut, and midgut is consistent with the dilatation of the ER tubules observed in these sections, although they could be osmium deposits in vacuoles. The ER of the hindgut also appeared slightly dilated during fasting, but did not demonstrate the same osmium deposition as the upper intestine. The dilatation of the ER during fasting resulted in an appearance similar to that of ER in the chloride cells of the gills of freshwater-adapted guppies (Pisam, 1981). These cells are involved in the transport of ions from the water to the blood. The similarity in appearance suggests that the ER of both cell types has a similar function, and because the structure is different from that of enterocytes of feeding flounders, that this ER structure relates to the osmoregulatory function. It appears to be these constituents which remain active and react with osmium.
The function of the hindgut is not known, although Ferraris and Ahearn (1984) have suggested that it behaves as a scavenger in nutrient uptake. The foregut of fish is the major site of ion handling from ingested seawater (Kirsch and Meister, 1982; Sleet and Weber, 1982). During intermittent drinking, as occurs in nature, the processed seawater that reaches the midgut is isosmotic to the blood. However, all areas of the intestine appear to be equally capable of ion transport (Field et al., 1978; Sleet and Weber, 1982). These latter studies did not include the hindgut. The hindgut of fasting flounder was frequently observed to be dilated with fluid, and the lumen often contained clear, crystalline material of unknown composition. It is likely, therefore, that the hindgut does not function in water uptake, but may be relatively impermeable, or even secrete fluid. The presence of lamellar structures, which are associated with ion and water transport (Yamamoto, 1966; Noailliac-Depeyre and Gas, 19761, suggests that secretion may be involved. The spectacular disappearance of ER impregnation with fasting in cells of the foregut and the midgut suggests that osmium uptake is related to the functional status of the ER. This was also suggested by preliminary studies in fasting for a short duration fasting rats. Interestingly, in jejunum of these fasting rats, cells of the top of the villous and of the crypt were the most affected. The fact that most of the cells of the mid-
FASTING-INDUCED MODIFICATION OF ER
Fig. 7.
Pyloric caecum, osmium impregnation. A. Feeding.
x 13,000.B: Fasting. x 10,500. Enterocytes from feeding fish showed
heavy impregnation in the large tubules of the ER. Fenestrated
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saccules were not observed. Impregnation was much reduced in fasting fish. Note the height of microvilli which were reduced in fasting conditions. Cell height was not diminished.
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Fig. 8. Foregut, osmium impregnation. A: Feeding. x 8,000. B: Fasting. x 8,000. Osmium impregnation of ER from feeding fish was again heavy, with heavily impregnated tubules as in the caeca, but a finer, extensive network as well. ER of fasting fish was poorly impregnated, with only dilated cisternae (or vacuoles) showing osmium black deposits.
FASTING-INDUCED MODIFICATION OF ER
Fig. 9. Midgut, osmium impregnation. A Feeding. x 8,000. Inset: x 11,000.B Fasting. x 5,000. The ER in feeding fish shows a fine network, with large tubules only at the level of the nucleus, probably Golgi apparatus. ER impregnation was reduced, but the Golgi remained impregnated, in fasting fish.
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Fig. 10. Hindgut, osmium impregnation. A Feeding. x 7,000. B Fasting. x 6,500. Osmium impregnation occurred only in large, discrete tubules in enterocytes from feeding fish. No ER network was observed. Impregnation was much reduced during fasting.
FASTING-INDUCED MODIFICATION OF ER
A
LEUCINE AMINOPEPTIDASE 601
FEEDING
B
ACKNOWLEDGMENTS
PROTEIN
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FOREGUT
MIDGUT
MINDGUT
J MAX CAECA
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The authors gratefully acknowledge the expertise and efforts of Mr. Jacques Bernier in the preparation of the photographs. They are also grateful to Mrs. Christiane Laurier for the secretarial work and to Drs. Georges Thiery and Jacques Paiement for their critical reading of the manuscript. The authors also acknowledge the skillful assistance of the Marine Research Laboratory (St. Andrew by the Sea), New Brunswick, Canada. This research was supported by grant MT-2862 of the Medical Research Council of Canada.
REFERENCES
10 0
c
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15
a
15 a
15
a
15
a
Fig. 11. A Leucine aminopeptidase activity measured at 8°C (mean 2 SE, n = 3). No significant differences were found between feeding or fasting fish, suggesting there is no change in enzyme quantity. B: Protein concentration showed a significant decline only in the hindgut (Mean +- SE, n = 3). C Maximal uptake rate (Jmax) of 14C-phenylalanine in enterocytes from fasting and feeding flounder, measured in vitro in mucosal sheets at 15°C and 8°C (Mean t SE, n = 5 or 6). Jmax was never significantly lower in fasting compared to feeding fish. These results icdicate that transporter concentration does not decrease during fasting. *Significantly different, P < 0.05.
villous were all impregnated is intriguing. We could speculate that these cells, being younger than those of the top of the villous, are less affected by fasting. The lack of impregnation might reflect the absence of certain molecules, such as amino acids which specifically react with osmium tetroxide (Thiery and Bergeron, 1984), when nutrient transport is abolished. The structural and chemical changes in the ER from winter flounder intestinal cells reflect the change in function of the intestine from feeding to fasting. These observations support a role for the ER in nutrient transport, but the nature of that role remains to be defined. They further confirm that osmium impregnation is a histichemical test of ER functions.
Appel, W. (1974) Amino acid arylamidases (''leucine nitroanilidase"). In: Methods in Enzymatic Analysis, Vol. 2. H.U. Bergmeyer, ed. Academic Press, New York, pp. 958-963. Bayerdorffer, E., Streb, H., Eckhardt, L., Haase, W., and Schulz, I. (1984) Characterization of calcium uptake into rough endoplasmic reticulum of rat pancreas. J. Membr. Biol., 81:69-82. Beaudry-Lonergan, M., Thiery, G., and Bergeron, M. (1985) Osmium impregnation of the endoplasmic reticulum correlates with the functional status of the prostatic secretory cell. Biol. Cell, 54:181186. Bergeron, M., and Thiery, G. (1981) Three-dimensional characteristics of the endoplasmic reticulum of rat renal tubule cells, an electron microscopy study in thick sections. Biol. Cell, 4243-48. Bergeron, M., Guerette, D., Forget, J., and Thiery, G. (1978) Threedimensional characteristics of the endoplasmic reticulum of the nephron a transcellular route. Kidney. Int., 13:102A. Bergeron, M., Gaffiero, P., and Thiery, G. (1987) Segmental variations in the organization of the endoplasmic reticulum of the rat nephron. A stereomicroscopic study. Cell Tissue Res., 247:215-225. Bergeron, M., Gaffiero, P., Berthelet, F. and Thiery, G. (1988) Interrelationship between organelles in kidney cells of adult and developing rat. Pediatr. Nephrol. 2:lOO-107. Berridge, M. (1984) Inositol trisphosphate and diacylglycerol as second messenger. Biochem. J., 220:345-360. Berthelet, F., Beaudry-Lonergan, M., and Bergeron, M. (1985) Proliferation of the endoplasmic reticulum of the proximal nephron cells during chronic metabolic acidosis and after treatment with triamcinolone. In: Biochemical Aspects of Kidney Function. 2. Kovacevic and W.G. Guder, eds. Walter de Gupter & Co. Berlin New York, pp. 213-219. Berthelet, F., Beaudry-Lonergan, M., Linares, H., Whittembury, G., and Bergeron, M. (1987) Polymorphic organization of the endoplasmic reticulum of the Malpighian tubule. Evidence for a transcellular route. La Cellule, 74:281-289. Ericsson, J.L.E. (1964) Absorption and decomposition of homologous hemoglobin in proximal tubular cells. An experimental light and electron microscopic study. Acta Pathol. Microbiol. Immunol. Scand [S~ppl.],168~1-121. Erkocak, R., Guathier, A., and Bucher, 0. (1963) A propos des modifications ultrastructurales dans les tubes initiaux du rein lors du diabete alloxanique. Ges. Exp. Med., 137:321-330. Ferraris, R.P., and Ahearn, G. (1984) Sugar and amino acid transport in fish intestine. Comp. Biochem. Physiol. [A], 77:397-413. Fletcher, G.L., Kiceniuk, J.W., and Williams, U.P. (1981) Effects of oiled sediments on mortality, feeding and growth of winter flounder, Pseudopleuronectes americanus. Mar. Ecol. Prog. Ser., 41914196. Gaffiero, P., Bergeron, M., and Thiery, G. (1983) Morphological study of cell organelles during development. I. The nuclear sac and the endoplasmic reticulum on the rat nephron. Biol. Cell, 49:79-82. Jard, S. (1981) Les isorecepteurs de la vasopressine dans le foie et dans le rein: Relation entre fixation d'hormone et reponse biologique. J. Physiol. (Paris), 77:621-628. Kennedy, V.S., and Steele, D.H. (1971) The winter flounder (Pseudopleuronectes americanus) in Long Pond, Conception Bay, Newfoundland. J. Fish. Res. Bd. Canada, 28:1153-1165. Kirsch, R., and Meister, M.F. (1982) Progressive processing of ingested water in the gut of sea-water teleosts. J. Exp. Biol., 98:6781
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Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193:265-275. Lukie, B.E., Westergaard, H., and Dietschy, J.M. (1974) Validation of a technique that allows measurement of both tissue uptake rates and unstirred layer thicknesses in the intestine under conditions of controlled stirring. Gastroenterology, 67:652-661. Madara, J.L., and Trier, J.S. (1987) Functional morphology of the mucosa of the small intestine. In: Physiology of the Gastrointestinal Tract, Volume 2. L.R. Johnson, ed. Raven Press, New York, pp. 1209-1249. McLeese, J.M. (1986) Seasonal changes in uptake of L-phenylalanine by the intestine of winter flounder, Pseudopleuronectes americanus. Ph.D. Thesis, University of Ottawa, Ottawa, Ontario, Canada. McLeese, J.M., and Moon, T.W. (1989) Seasonal changes in the intestinal mucosa of winter flounder, Pseudopleuronectes americanus (Walbaum), from Passamaquoddy Bay, New Brunswick. J . Fish Biol., In press. Mollgard, K., and Rostgaard, J . (1978) Morphological aspects of some transporting epithelia suggesting ‘a transcellular pathway via elements of endoplasmic reticulum. J. Membr. Biol., 40:71-89. Muallem, S., Schoeffield, M., Pandol, S., and Sacks, G. (1985) Inositol trisphosphate modification of ion transport in rough endoplasmic reticulum. Proc. Natl. Acad. Sci. U.S.A., 82:4433-4437. Noailliac-Depeyre, J . , and Gas, N. (1976) Electron microscopic study on gut epithelium of the tench (Tinca tinca L.) with respect to its absorptive function. Tissue Cell, 8511-530. Nonnotte, L., Nonnotte, G., and Leray, C. (1986) Morphological changes in the middle intestine of the rainbow trout, Salmo gairdnerr, induced by a hyperosmotic environment. Cell Tissue Res., 243: 619-628. Nordlie, R.C. (19791 Multifunctional glucose-6-phosphatase: Cellular biology. Life Sci., 24:2397-2404. Novikoff, A.B., Spater, H.W., and Quintana, N. (1983) Transepithelial
endoplasmic reticulum in rat proximal convoluted tubule. J . Histochem. Cytochem., 31:656-661. Palade, G.E. (1955) Studies on the endoplasmic reticulum. 11. Simple dispositions in cells in situ. J . Biophys. Biochem. Cytol., 1567-582. Porter, K.R. (1953) Observations on a submicroscopic basophilic components of cytoplasm. J . Exp. Med., 97:727-749. Porter, K.R., and Palade, G.E. (1957) Studies on the endoplasmic reticulum. 111. Its form and distribution in striated muscle cells. J. Biophys. Biochem. Cytol., 3:269-299. Pisam, M. (1981) Membranous systems in the “chloride cell” of teleostean fish gill; their modifications in response to the salinity of the environment. Anat. Rec., 200:401-414. Skadhauge, E., and Lotan, R. (1974) Drinking rate and oxygen consumption in the euryhaline teleost Aphanius dispar in waters of high salinity. J . Exp. Biol., 60547-556. Sleet, R.B., and Weber, L.J. (1982) The rate and manner of seawater ingestion by a marine teleost and corresponding modification by the gut. Comp. Biochem. Physiol. [A], 72:469-475. Thiery, G. (1979) Colorations signaletiques sur coupes epaisses du reticulum endoplasmique, de la chromatine et des surfaces cellulaires libres des cellules animales. Biol. Cell, 35159-164. Thiery, G., and Rambourg, A. (1976) A new staining technique for studying thick sections in the electron microscope. J. Microsc. Biol. Cell., 26:103-106. Thiery, G., and Bergeron, M. (1984) Signification de la coloration du RE des cellules animales par impregnation osmique. Biol. Cell, 51: 31a. Thiery, G., Gaffiero, P., and Bergeron, M. (1983) Three-dimensional characteristics of the endoplasmic reticulum in columnar cells of the rat small intestine. An electron microscopy study in thick sections. Am. J . Anat., 167:479-493. Yamamoto, T. (19661 An electron microscopic study of the columnar epithelial cell in the intestine of fresh water teleosts: Goldfish (Carassius auratus) and rainbow trout (Salmo gairdneri). Z. Zellforsch., 72:66-87.
Fasting Induces Modifications of the Endoplasmic Reticulum in Intestinal Cells JENNIFER McLEESE AND MICHEL BERGERON Department of Physiology, UniuersitQde Montrkal, Montrkal H3C 357, QuQbec,Canada
KEY WORDS
Epithelial cells, Transport, Intestinal cell, Flounder
ABSTRACT
The polymorphism of the endoplasmic reticulum (ER) in epithelial cells with different transport functions such as the enterocyte suggests that the ER may be involved in some way in molecular transport. To further access this possibility, we examined the ER from the intestine of winter flounder, Pseudopleuronectes americanus, a species which undergoes an annual fast of approximately 6 months’ duration, a time during which previous work indicates nutrient carrier number does not change. Fish from June (feeding) and January (8-10 weeks fasted) were sampled. Tissues from the pyloric caeca, foregut, midgut, and hindgut were prepared for electron microscopy using two techniques of staining. Cell height was unaltered in any section, although microvillar length shortened variably. Cellular organization, including position of nuclei, number and distribution of mitochondria, and presence of basolateral membranes, did not change. The ER appeared equally abundant in June and January. However, use of the osmium impregnation technique, which is specific for ER cisternal contents, revealed a change in the impregnation of ER, from a heavily impregnated network in summer t o little or no impregnation in winter. These results suggest that a shift in function of the ER had occurred when nutrient transport ceased, and supports arole of the ER in nutrient transport.
INTRODUCTION
metal impregnation technique (Bergeron and Thiery, 1981; Bergeron et al., 1978; Mollgard and Rostgaard, The extensive organization of the ER in transport 1978; Thiery et al., 1983) as well as other histochemical epithelial cells and its polymorphic characteristics techniques (Novikoff et al., 19831, offers a logical morhave been underestimated if not ignored. In any model phological basis for the integrated functions that the of transport epithelial cell, the plasma membranes, RER, SER, and Golgi apparatus appear to carry out in junctional complexes, and mitochondria are always absorptive cells (see for review Madara and Trier, portrayed, whereas the ER is seldom, if ever, repre- 1987). sented. Yet, in most epithelial cells the ER is a wellThe ER is portrayed, almost exclusively, as the site organized network (Bergeron and Thiery, 1981; Berg- of protein synthesis and its schematic representation eron et al., 1978, 1987, 1988; Berthelet et al., 1987; follows Palade’s concepts based on the pancreatic aciMollgard and Rostgaard, 1978; Novikoff et al., 1983; nar cells, a large producer of proteins. However, all Palade, 1955; Porter, 1953; Porter and Palade, 1957; eukaryotic cells have an ER and a Golgi apparatus, yet Thiery 1979; Thiery et al., 1983). In both the rat jeju- protein secretion is not their primary function. Renum and proximal nephron, the ER organization is al- cently the ER was found to be a reservoir for calcium most identical: a continuous network of canaliculi and ions which are liberated following hormonal or pharfenestrated saccules extending from the apex, below macological stimuli which initiate the phosphoinositol the microvilli, to the lateral and basilar plasma mem- cascade a t the V, receptors (Bayerdorffer et al., 1984; branes and sending many projections to the nuclear Berridge, 1984; Muallem et al., 1985). The absence of envelope. Circular, or at times flattened, canaliculi of both V1 receptors (Jard, 1981) and of a well-organized irregular diameters varying between 0.005-0.03 FM, ER (Bergeron et al., 1987) in principal cells of the renal run more-or-less parallel to the long axis of the cell. collecting tubules is an intriguing finding since calFenestrated saccules, possessing fairly regular perfo- cium liberation from the ER is crucial in the inositol rations, averaging 0.03 FM, are interposed on the path pathway. of the canaliculi; these fenestrated saccules surround In epithelial cells, the ER is also a key element in the apical vacuoles and mitochondria as well as cytoplas- regulation of transport carriers and their insertion into mic bodies and vary accordingly in size. The nuclear envelope appears as a specialized part of the ER, which also seems, in thick sections, to be connected to the Golgi apparatus. Along the lateral plasma membranes extensive fenestrated saccules are observed and were Received July 31, 1989; accepted in revised form August 30, 1989 in fact previously described in ultrathin sections as the Address reprint requests to Michel Bergeron, M.D., Departement de physiolparamembranous cisternal system (Ericsson, 1964; ogie, Universit6 de MontrBal, C.P. 6128, Succursale A, Montreal H3C 357, Qu& Erkocak et al., 1963).This ER continuity, shown by the bec. Canada.
0 1990 WILEY-LISS, INC
FASTING-INDUCED MODIFICATION OF ER
basolateral or apical membranes. The fact that the ER appears, in three-dimensional photographs, to constitute a transcytoplasmic route led some investigators (Mollgard and Rostgaard, 1978) to suggest that it may constitute an additional transport system for sodium, a highly questionable hypothesis. Moreover, the ER is not a purely static cellular compartment but shows extensive variations in its osmium staining properties. For example, the evolution of the ER during cellular ascent from the intestinal crypt to the top of the villus is towards a greater complexity (Thiery et al., 1983). At the base of the crypt of Lieberkun, no definite organization is noted; and, most often, only the nuclear envelope and a few canalicular elements are seen. In the mid-crypt, the ER becomes organized, and fenestrated saccules appear and in mature columnar cells the ER finally forms, a transcytoplasmic continuous network of canaliculi and fenestrated saccules. Such an evolution of the ER organization is also noted during renal ontogeny (Gaffiero et al., 1983). The ER impregnation was found to correlate with their physiological status. It can be modified by hormonal factors in prostatic secretory cells (BeaudryLonergan et al., 19851,by chronic metabolic acidosis, or treatment with triamcinolone in proximal tubule cells of the rat kidney (Berthelet et al., 1985). These data suggested that ER impregnation is related to cellular activity and that this technique may provide a test for cell function and organelle interrelations. In jejunal cells, the question arises as to whether ER impregnation is somehow related t o the absorption of nutrients; could the ER function as a transcellular route for movement of molecules such as peptides, amino acids, or glucose (Bergeron and Thiery, 1981; Bergeron et al., 1978; Nordlie, 1979) or sodium (Mollgard and Rostgaard, 1978). In jejunal villi of well-fed rats, cells are generally well-impregnated, but it is not uncommon to see cells with a non-impregnated ER, a phenomenon which appears to be amplified in fasting rats. Because of ethical considerations it is difficult to obtain a definite answer in fasting mammals, and we have therefore examined the ER from the intestine of winter flounder, Pseudopleuronectes americanus, a species which undergoes a natural annual fast of approximately 6 months' duration, a time during which previous work indicates nutrient carrier number per unit surface area does not change (McLeese, 1986). Our results revealed a change in the osmium impregnation of ER, from a heavily impregnated network in summer (feeding) to little or no impregnation in winter (fasting). These results in fishes as well as fragmentary results in the rat suggest that a shift in function of the ER has occurred when nutrient transport ceased, and indirectly support a role of the ER in nutrient transport. MATERIALS AND METHODS Winter flounders were caught from Passamaquoddy Bay (New Brunswick, Canada) by otter trawl and brought to the Huntsman marine laboratory where they were maintained in tanks of running seawater. Fish sampled in the summer were used within 1day of
57
capture; fish for winter sampling were brought to the laboratory in October and held until early January. They were not offered food through this holding period, which corresponds to a natural fasting time (Fletcher et al., 1981; Kennedy and Steele, 1971). Fish were sacrificed by cervical dislocation. The intestines were rapidly excised and segments from the pyloric caeca, foregut, midgut, and hindgut were removed and placed in fixative. Tissues were subsequently prepared by one of two methods: the osmium impregnation technique of Thiery (Thiery, 1979; Thibry et al., 1983) or the lead and copper citrate technique of Thiery and Rambourg (1976).Briefly, osmium impregnation, which stains the cisternal contents of the endoplasmic reticulum, involves fixation in 2.5% glutaraldehyde for 20 min, followed by postfixation in 1%osmium tetroxide for 1.5 hr a t 4°C. Tissue blocks were then left in 1%osmium tetroxide a t 37°C for 3 days. The osmium solution was replaced daily. The lead and copper citrate technique requires fixation in 2.5% glutaraldehyde. Leucine aminopeptidase activity was assessed a t 8°C by the method of Appel(1974). Protein was determined by the method of Lowry et al. (1951). Phenylalanine uptake by emusculated sheets of intestinal mucosa was measured with '*C-radiolabelled phenylalanine using a system based on that described by Lukie et al. (1974). Incubation times was 2 min, followed by a 20 sec rinse in unlabelled flounder Ringer's. Tissue was cut free of the mounting chamber, dried to determine dry weight, then solubilized and counted for radioactivity by liquid scintillation counting. Tritiated mannitol was employed as the unabsorbed marker.
RESULTS Preliminary data have shown that the ER osmium impregnation of jejunal cells was modified by fasting in rats. Figure 1 shows the intensive impregnation of the ER and its rich organization as a network, which was previously described (Thiery et al., 1983). Four days after food was removed, which corresponds to a fasting of about 24 hours (or more), almost all jejunum cells in the mid-crypt still have a well-impregnated ER (Fig. l), while most cells located at the top of the villous are not impregnated with osmium (Fig. 2). Such a striking difference from the well-fed rats may imply that ER has a role in nutrient transport. Because of ethical considerations, we have looked for a species which undergoes a natural fast. We have therefore examined the ER of gut cells of winter flounders, Pseudopleuronectus americanus. Figure 3 is a schematic representation illustrating the various functional and anatomical divisions of the flounder gut, which is much less specialized than the mammalian gut. The gut was arbitrarily divided into four sections, but these were found to be functionally different (McLeese, 1986). Enterocytes of the flounder were tall and narrow. Mitochondria and endoplasmic reticulum (ER) were abundant in the cell apex, but were also present basally (Figs. 4A, 5A). A prominent Golgi apparatus was usually supranuclear. The nucleus was located toward the basal half of the cell, and the nuclear envelope was
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Fig. 1. Mid-crypt of the rat jejunum. Note the extensive ER organization revealed by osmium impregnation after 5 days. Under the brush border, one sees a continuous network of canaliculi and fenestrated saccules which are themselves conected to other canaliculi. Fenestrated saccules are surrounding the mitochondria. The perinuclear membrane is well stained and the pores can be easily seen in
some nuclei (A). Canaliculi are in contact with the nuclear membrane. Note that, in this fasting animal (4 days), all cells of the mid villous are impregnated with osmium. Fasting seems to induce intracellular dilatation. (For further description of the ER of the mammalian jejunum enterocyte, see Thi6ry et al., 1983.) x 2,000.
observed at times to be continuous with the elements of the ER. Microvilli were present at the luminal face of the cells, and intermicrovillous invaginations were observed in all segments of the intestine. Numerous small vacuoles were present immediately beneath the microvilli and in the apical cytoplasm. Microvesicular bodies were common apically in all segments. Lamellar structures, which resembled the lateral plasma membranes in their staining properties, were observed in all segments of the intestine. They are analogous to the basilar infolding seen in many transport epithelia. They were especially abundant basally, but were also present apically in many cells. In many cases, ER cisternae were in close association with mitochondria (Fig. 6). After 8-10 weeks of fasting, the enterocytes did not appear to be shortened, although microvilli were variably shortened and sometimes reduced in number (Figs. 4B,5B). The alterations in the microvillar morphology did not appear to be consistent: in a given region of the gut, adjacent cells showed short and long microvilli. Mitochondria1 profiles were less numerous apically. Microvesicular bodies and intermicrovillous invaginations were still seen in many cells, particularly those in the hindgut. Dense bodies were present in the apical cytoplasm in all segments. Such struc-
tures were not observed in the enterocytes of feeding fish. The lamellar structures of the basilar plasma membranes appeared unchanged by feeding condition. Apical ER appeared to have become more dilated with fasting (Fig. 5B). Large, irregularly shaped cisternae were prominent in many cells, particularly those of the caeca and the foregut (Fig. 6).
Osmium impregnation (Figs. 7-10) Osmium impregnation specifically stains the cisternal contents of the ER. Cut in thick sections, osmium impregnated tissues give an idea of the three dimensional structure of the ER. Fenestrated saccules such as are observed in mammalian intestine (Thiery et al., 1983) were not present in any segment (Figs. 7-10). Impregnation of ER tubules, not associated with the nucleus, differed markedly according to feeding status, and the tubules displayed a high degree of regional polymorphism (Figs. 7-10). The apical ER of the pyloric caeca from feeding flounder could be discerned as large, discrete tubules heavily impregnated with osmium (Fig. 8A). Large, heavily impregnated regions, almost saccules, as well as smaller, fine networks of tubules were observed in the foregut (Fig. 8A). In the midgut, fine, complex networks were observed (Fig. 9A), whereas only large sac-
FASTING-INDUCED MODIFICATION OF ER
59
Fig. 2. Top of the villous of the rat jejunum. Fasting for 4 days. Note that most cells are poorly or not impregnated. One cell shows the extensive normal ER network seen in normal fed animals. Most Golgi are well-impregnated (arrows). Osmium impregnation, x 2,000.
cosal protein concentrations were significantly lower in fasting fish than in feeding fish only in the hindgut.
DIVISIONS OF THE FLOUNDER INTESTINE STOMACH
WLORIC SPHINCTER
Fig. 3.
FOREGUT
PYLORIC CAECA
MIDGUT
HINOGUT
VALVE
Schematic representation of the flounder gut.
cules of osmium deposits were observed in the hindgut (Fig. lOA), without smaller tubules. In fact, some cells had a well-developed ER similar to the rat jejunum. The more basal regions were similar regardless of region, with large deposits of osmium rather than tubular networks. Osmium deposition was much reduced in cells from fasting fish. Fine networks were never observed. However, large deposits occurred in all segments except the hindgut. Osmium impregnation was least reduced by fasting in the foregut.
Physiological characteristics Neither the maximal uptake rate (Jmax) of phenylalanine nor the activity of leucine aminopeptidase decreased in any segment following fasting (Fig. ll).Mu-
DISCUSSION The intestinal mucosa of animals is well known to atrophy disproportionately to the body during starvation. This is thought to be a result of the loss of direct stimulation by nutrients. However, the intestine of a marine fish serves a dual function, acting as an important organ of osmoregulation as well as of digestion. In the winter flounder, the annual winter fast results in a change of function from digestion and osmoregulation to osmoregulation alone (Nonnotte et al., 1986; Skadhauge and Lotan, 1974; Sleet and Weber, 1982). Ion and water transport for osmoregulation has been suggested to ameliorate the effects of fasting on the surface area of the intestine in this fish, and help in the maintenance of the proximodistal gradient of surface area (McLeese and Moon, 1989). The results from the leucine aminopeptidase assays and the phenylalanine uptake experiments suggest that no decrease in enzyme or carrier number per surface area occurred with fasting. Because turnover times for these membrane components are not known, it is difficult to assess whether protein synthesis continued during fasting. However, since protein concentration decreased only in the hindgut, and it was only in the hindgut that osmium impregnation was totally
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Fig. 4. Hindgut of the flounder. Staining with lead and copper citrate and uranyl acetate. A: Feeding. x 20,000. B: Fasting. X 8,500.The ER was again more dilated in B than in A (arrows). Note the microvilli which are smaller in fasting animals than in well-fed flounders.
FASTING-INDUCED MODIFICATION OF ER
Fig. 5. Pyloric caecum of the flounder. Staining with lead and copper citrate and uranyl acetate. A: Feeding. x 13,000. B: Fasting. x 13,000. The ER network is extensive and cisternae are dilated after fasting (arrows). Some cisternae have the aspect of big vacuoles.
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Fig. 6. Foregut of feeding fish. Staining as in Figures 4 and 5 . Photograph showing plasma membranes and extensive ER dilatations. Note the close association between mitochondria and ER, which was described in rat jejunum and proximal nephron. (See Thiery et al., 1983, for further description of organelle relationship.) x 28,000.
abolished, it seems likely that the ER was still active to some extent in the other three intestinal sections. Beaudry-Lonergan et al. (1985) demonstrated a change in the structure and chemistry (indicated by the altered osmium black deposition) of the ER with altered function of the rat prostatic secretory cell. The present study suggests that analogous changes occurred in the enterocytes of the winter flounder intestine. As seen from Figure 4B, the ER of the upper intestine became more dilated during fasting. The appearance of large deposits of osmium black in the caeca, foregut, and midgut is consistent with the dilatation of the ER tubules observed in these sections, although they could be osmium deposits in vacuoles. The ER of the hindgut also appeared slightly dilated during fasting, but did not demonstrate the same osmium deposition as the upper intestine. The dilatation of the ER during fasting resulted in an appearance similar to that of ER in the chloride cells of the gills of freshwater-adapted guppies (Pisam, 1981). These cells are involved in the transport of ions from the water to the blood. The similarity in appearance suggests that the ER of both cell types has a similar function, and because the structure is different from that of enterocytes of feeding flounders, that this ER structure relates to the osmoregulatory function. It appears to be these constituents which remain active and react with osmium.
The function of the hindgut is not known, although Ferraris and Ahearn (1984) have suggested that it behaves as a scavenger in nutrient uptake. The foregut of fish is the major site of ion handling from ingested seawater (Kirsch and Meister, 1982; Sleet and Weber, 1982). During intermittent drinking, as occurs in nature, the processed seawater that reaches the midgut is isosmotic to the blood. However, all areas of the intestine appear to be equally capable of ion transport (Field et al., 1978; Sleet and Weber, 1982). These latter studies did not include the hindgut. The hindgut of fasting flounder was frequently observed to be dilated with fluid, and the lumen often contained clear, crystalline material of unknown composition. It is likely, therefore, that the hindgut does not function in water uptake, but may be relatively impermeable, or even secrete fluid. The presence of lamellar structures, which are associated with ion and water transport (Yamamoto, 1966; Noailliac-Depeyre and Gas, 19761, suggests that secretion may be involved. The spectacular disappearance of ER impregnation with fasting in cells of the foregut and the midgut suggests that osmium uptake is related to the functional status of the ER. This was also suggested by preliminary studies in fasting for a short duration fasting rats. Interestingly, in jejunum of these fasting rats, cells of the top of the villous and of the crypt were the most affected. The fact that most of the cells of the mid-
FASTING-INDUCED MODIFICATION OF ER
Fig. 7.
Pyloric caecum, osmium impregnation. A. Feeding.
x 13,000.B: Fasting. x 10,500. Enterocytes from feeding fish showed
heavy impregnation in the large tubules of the ER. Fenestrated
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saccules were not observed. Impregnation was much reduced in fasting fish. Note the height of microvilli which were reduced in fasting conditions. Cell height was not diminished.
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Fig. 8. Foregut, osmium impregnation. A: Feeding. x 8,000. B: Fasting. x 8,000. Osmium impregnation of ER from feeding fish was again heavy, with heavily impregnated tubules as in the caeca, but a finer, extensive network as well. ER of fasting fish was poorly impregnated, with only dilated cisternae (or vacuoles) showing osmium black deposits.
FASTING-INDUCED MODIFICATION OF ER
Fig. 9. Midgut, osmium impregnation. A Feeding. x 8,000. Inset: x 11,000.B Fasting. x 5,000. The ER in feeding fish shows a fine network, with large tubules only at the level of the nucleus, probably Golgi apparatus. ER impregnation was reduced, but the Golgi remained impregnated, in fasting fish.
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Fig. 10. Hindgut, osmium impregnation. A Feeding. x 7,000. B Fasting. x 6,500. Osmium impregnation occurred only in large, discrete tubules in enterocytes from feeding fish. No ER network was observed. Impregnation was much reduced during fasting.
FASTING-INDUCED MODIFICATION OF ER
A
LEUCINE AMINOPEPTIDASE 601
FEEDING
B
ACKNOWLEDGMENTS
PROTEIN
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The authors gratefully acknowledge the expertise and efforts of Mr. Jacques Bernier in the preparation of the photographs. They are also grateful to Mrs. Christiane Laurier for the secretarial work and to Drs. Georges Thiery and Jacques Paiement for their critical reading of the manuscript. The authors also acknowledge the skillful assistance of the Marine Research Laboratory (St. Andrew by the Sea), New Brunswick, Canada. This research was supported by grant MT-2862 of the Medical Research Council of Canada.
REFERENCES
10 0
c
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15
a
15 a
15
a
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a
Fig. 11. A Leucine aminopeptidase activity measured at 8°C (mean 2 SE, n = 3). No significant differences were found between feeding or fasting fish, suggesting there is no change in enzyme quantity. B: Protein concentration showed a significant decline only in the hindgut (Mean +- SE, n = 3). C Maximal uptake rate (Jmax) of 14C-phenylalanine in enterocytes from fasting and feeding flounder, measured in vitro in mucosal sheets at 15°C and 8°C (Mean t SE, n = 5 or 6). Jmax was never significantly lower in fasting compared to feeding fish. These results icdicate that transporter concentration does not decrease during fasting. *Significantly different, P < 0.05.
villous were all impregnated is intriguing. We could speculate that these cells, being younger than those of the top of the villous, are less affected by fasting. The lack of impregnation might reflect the absence of certain molecules, such as amino acids which specifically react with osmium tetroxide (Thiery and Bergeron, 1984), when nutrient transport is abolished. The structural and chemical changes in the ER from winter flounder intestinal cells reflect the change in function of the intestine from feeding to fasting. These observations support a role for the ER in nutrient transport, but the nature of that role remains to be defined. They further confirm that osmium impregnation is a histichemical test of ER functions.
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