© 1990 S. Karger AG, Basel 0006-3126/90/0582-010452.75/0
Biol Neonate 1990;58:104-111
Intestinal Absorption of Macromolecules and Epithelial Cell Surface Glycosylation in Suckling Rats Nursed on Mothers Fed Low-Protein Diet-I HarjitS. Babbar, Virender M.S. Jaswal, Reena Gupta, Akhtar Mahmood Department of Biochemistry, Panjab University, Chandigarh, India
Key Words. Maternal nutrition • Macromolecular absorption • Membrane glycosylation • Lectin binding • Sialylation • Fucosylation
Introduction Surface oligosaccharides play a vital role in various cell functions; these include cell differentiation and recognition. They act as receptors for hormones, toxins and lectins and are involved in membrane transport processes [1-3]. The microvillus membrane (MVM) lining the enterocytes contains con
siderable amounts of carbohydrates [4], We have previously shown that surface-reactive carbohydrates, in particular sialic acid and fucose, undergo striking modifications dur ing postnatal development in rat intestine [5,6]. During early suckling period, the membrane is rich in sialic acid and contains low levels of fucose but the pattern is re versed on weaning [6], Biol et al. [7, 8] have
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Abstract. The effect of feeding 8 % protein (low-protein; LP) diet to lactating rats has been studied on the absorption of macromolecules and the glycosylation of enterocytes in suckling animals. The absorption of 125I-labelled bovine serum albumin, y-globulin and a-lactalbumin was 24-314 % more in pups nursed on mothers fed LP diet compared to the pair-fed controls. The observed enhancement in protein absorption was associated with a significant increase in the binding of these proteins to microvillus membranes (MVM). The sialic acid content of brush borders was unaltered but the fucose level was augmented (p < 0.01) in pups nursed on rats fed LP diet. The binding of 125I-labelled wheat germ agglutinin and Ulex europeus agglutinin I to MVM was in agreement to the data on sialic acid and fucose levels of the membranes. The binding of peanut agglutinin to MVM was 30% low in pups from LPdiet-fed animals. The incorporation of (l4C) Z)-mannose into MVM was essentially unaf fected but that of (l4C) N-acetylglucosamine was reduced in pups reared on mothers given LP diet. These findings suggest that the quality of maternal nutrition affects the absorption of macromolecules and the glycosylation in developing rat intestine.
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Maternal Nutrition and Intestinal Glycosylation
Materials and Methods Albino rats (Wistar strain) were used. Pregnant animals were fed on a stock diet (Hindustan Lever, Bombay, India) with free access to water. On day 1, postparturition, lactating rats were fed on LP diet (ta ble 1) for 16 days. Control animals were pair-fed. Ani mals in the normal-fed group were given a rat pellet diet (18% protein) ad libitum. There were 6-8 pups per mother and at least 4 litters of each group were employed. On day 17 after birth, overnight-fasted pups were sacrificed under light ether anesthesia. Starting from the ligament of Treitz, entire intestine was removed, thoroughly washed in ice-cold saline and used for various biochemical studies. Absorption o f 125¡-Labelled Proteins The absorption of 125I-bovine serum albumin (BSA), y-globulin and a-lactalbumin was studied in vivo [11]. 0.2 ml of phosphate-buffered saline (PBS), pH 7.2 containing 300-500 pg BSA or y-globulin or a-lactalbumin and trace amounts of l2SI-labelled pro teins (specific activity 3 X 104—5 X 105 cpm/pg pro tein) were administered orally to each rat (body weight 18-22 g) using Ryle’s tube. Animals were sac rificed after 2 h. Blood was collected directly from the heart in heparinized tubes. The intestinal tissue was washed with PBS, blotted, weighed and the radioac tivity was determined in y-counter. For measuring the absorption of proteins into blood, suitable volume of the blood was treated with trichloroacetic acid (TCA) to a final concentration of 10% and the tubes were left
Table 1. The composition of control and LP diets fed to lactating rats Ingredient
Control, g
LP, g
Casein Starch/sucrose Cellulose Com oil Salt mixture Vitamin mixture
18 50 14 10 6 2
8 63 11 10 6 2
100 g of each diet provides 380 calories.
overnight at 4 °C. The contents were centrifuged and the supernatant was removed. Precipitates were washed twice with 10% TCA and the radioactivity was determined. The absorption of proteins was ex pressed as pg of l25I-protein/ml of blood. Measurement o f Protein Absorption by ELISA Antibodies against BSA and y-globulin were raised in rabbits by injecting 1.5 mg i.m. of the pro tein along with 250 pi of Freund's complete adjuvant. After 3 weeks, the same amount of the protein mixed with incomplete adjuvant was injected into each ani mal, followed by two more injections with a gap of 15 days each. Blood was obtained by cardiac puncture, allowed to clot at room temperature and was centri fuged at 3,000 g for 10 min. Blood serum was re moved and stored at -2 0 ° C . The presence of anti bodies against BSA and y-globulin was detected by immunodiffusion, carried out in agarose plates. Immunoreactive BSA and y-globulin in the serum of pups from the control and LP groups was detected by ELISA, using radish-peroxidase-labelled antirabbit antibodies as described by McLean and Ash [ 12]. The absorption of proteins into blood was expressed as pg/ml blood. Preparation o f MVM Brush borders were isolated and purified follow ing the method of Kessler et al. [13]. The membranes, suspended in 50 mM sodium maleate buffer (pH 6.8), exhibited a 12- to 15-fold enrichment of brush-border sucrase and alkaline phosphatase activities.
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described that the quality as well as the quantity of diet affects the glycosyl-transferase levels in intestine. Since milk is the sole source of nutrition for the developing pups during the suckling period and the composi tion of milk is altered in response to changes in the maternal nutrition [9, 10], we studied the effect of feeding low-protein (LP) diet (8% protein) to lactating rats on the absorp tion of 125I-labelled proteins and the glycosy lation of intestinal microvillus membrane in suckling rats.
Babbar/Jaswal/Gupta/Mahmood
106
Fig. 1. Growth curve of suckling rats: control (A); pair-fed control (•) and LP groups (■). Bars indicate SD of the means.
immediately through 0.2 pm Millipore filter (EGWP, Milliporc Corporation. Bedford, Tex., USA) under suction. The filters were washed twice with 3 ml of PBS and the radioactivity retained on the filter was counted with a y-counter. Suitable controls to deter mine nonspecific protein binding were run simulta neously [6]. After correcting for the nonspecific bind ing, the protein/lectin binding to the membranes was expressed as pg of protein/lectin-bound/mg mem brane protein. Fucose content of the membranes was determined colorimetrically after initial hydrolysis of the membranes with 0.1 N H^SOj for 4 h at 100 °C [15]. Sialic acid was determined by the method of Skoza and Mohos [ 16] after digesting the membranes with 0.1 N H2SO4 for 60 min at 80 °C. Protein was determined according to Lowry et al. [17] using BSA as the standard. Statistical analysis of the data was done by Stu dent’s t test. All the reagents used were of analytical grade.
Labelling o f Proteins and Lectins with Various proteins and lectins were labelled with l25I as described earlier [14], 0.2 ml of reaction mix ture containing 100 pg protein or lectin, 0.3 mCi N al25I, 500 pg chloramine T and 0.2 M phosphate buffer (pH 7.2) was incubated at 30 °C. After 60 s. the reaction was terminated by adding 0.1 ml of sodium metabisulphite (lOmg/ml). Unreacted 125I was re moved by passing the mixture through Scphadex G 25 column (0.5 X 15 cm) followed by exhaustive dial ysis against 20 mA/Tris-HCl (pH 7.2). Specific activ ity of various labelled protcins/lcctins ranged between 2 X 104-5 X 10s cprn/pg protein. Binding o f¡ - L a b e lle d Proteins or Lectins to MVM The binding of BSA, y-globulin. a-lactalbumin and various lectins namely wheat germ agglutinin (WGA), Ulex europeus agglutinin (UEA[) and peanut agglutinin (PNA) to brush borders was measured as described previously [6]. In a total volume of 0.2 ml, 30 pg membrane protein was incubated with 2-4 pg of l25l-labelled protein or lectin in PBS (pH 7.2) at 30 °C for 30 min. Reaction was terminated by adding 4 ml of ice-cold PBS and the contents were filtered
As shown in figure 1, the gain in the body weight of pups weaned on dams given, LP diet was relatively small compared to that in the control group. At the time of sacrifice, the body weight of pups in the LP group was 35% low compared to controls. The intesti nal weight of suckling rats was also signifi cantly (p < 0.01) low in the LP group (0.47 ± 0.08 g) compared to control pups (0.82 ± 0.14 g). However, there was no difference in the intestinal length (39.2-41.4 cm) under these conditions. The intestinal tissue during the perinatal period exhibits high pinocytotic activity re sulting in the absorption of intact macromol ecules, and this process ceases on weaning [18], Thus, the absorption of l25I-BSA, yglobulin and a-lactalbumin was investigated in pups from control and experimental groups. The appearance of BSA in blood was 135% higher in the LP group compared to
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Results
Maternal Nutrition and Intestinal Glycosylation
107
Table 2. Effect of feeding LP diet to lactating mothers on the absorption of labelled protein in suckling rats Group
BSA1
y-Globulin1
a-Lactalbumin1
Ad-libitum-fed control
TCA precipitate ELISA
1.33 ± 0.58 0.50 ±0.08
0.11 ±0.01 0.04 ±0.01
0.40 ±0.01
Pair-fed control
TCA precipitate ELISA
1.07 ±0.70 0.41 ±0.12
0.07±0.01 0.03 ±0.01
0.55 ±0.03
LP Fed
TCA precipitate ELISA
2.51 ±0.69* 0.92±0.10**
0.29 ±0.02** 0.16 ±0.01**
0.68 ±0.05**
the pair-fed controls (table 2). Nearly a 4fold increase in y-globulin and a significant (p < 0.001) elevation in a-lactalbumin ab sorption was observed under these condi tions. Measurement of BSA and y-globulin in the serum by ELISA also revealed a marked increase (p < 0.001) in the absorp tion of these proteins in pups nursed on lac tating rats fed LP diet. Still, the uptake val ues obtained by the TCA precipitation method were considerably higher compared to those obtained by ELISA. Such a differ ence in the detection of serum proteins by TCA precipitation and immunodiffusion methods has earlier been reported by Udall et al. [11]. Analysis of l25I-labelled proteins in intestinal tissue also revealed a significant increase in the protein uptake in pups from the LP group compared to controls (results not shown). This indicated that both the tis sue uptake of proteins and their absorption into blood was augmented in pups nursed on rats fed LP diet. In has been shown that in vitro uptake of BSA, y-globulin and a-lactalbumin follows a saturation phenomenon suggesting the in
volvement of a receptor-mediated process for their uptake [14]. Therefore, the binding of 125I-labelled proteins to MVM was also examined under these conditions. As shown in table 3, the binding of BSA, y-globulin and a-lactalbumin to MVM was 20-37% higher in the LP group compared to the pair-fed control. Thus, the observed increase in the absorption of proteins in suckling rats is pre sumably a consequence of enhanced binding of the macromolecules to the epithelial cell surface in response to LP diet given to the lactating rats. It may also be pointed out that there was essentially no difference in body weight, absorption of proteins, binding data and membrane glycosylation between ad li bitum controls and pair-fed groups. There fore, the results obtained from pups weaned on dams given LP diet were compared with those from the pair-fed animals, hence re ferred as ‘controls’. Sialic acid and fucose are interesting sug ars, which generally occupy the nonreducing terminal ends and thus act as stoppers of oligosaccharidic chains in glycopeptides [1,2], Analysis of these sugars in brush bor-
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Values are mean ± SD of 4-6 observations. * p < 0.05 and ** p < 0.001 compared to pair-fed controls. 1 pg protein absorbed/ml blood.
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Babbar/Jaswal/Gupta/Mahmood
Table 3. Binding of l25I-labelled proteins to microvillus membranes from suckling rats nursed on mothers fed LP diet Group
BSA1
y-Globulin1
a-Lactalbumin1
Ad-libitum-fed control Pair-fed control LP fed
2.4±0.2 1.9 ±0.4 3.1 ±0.1**
5.5 ±0.6 6.1 ±0.2 9.2± 1.5**
6.2±0.2 6.7±0.2 8.4±0.5*
Values are mean ± SD of 4-6 observations. * p < 0.05 and ** p < 0.01 compared to pair-fed controls. 1 pg protein bound/mg protein.
Table 4. The binding of l25l-labelled WGA, UEAi and PNA to MVM of suckling animals nursed on mothers fed LP diet Group
Sialic acid1
Fucose1
WGA2
UEAi2
PNA2
Ad-libitum-fed control Pair-fed control LP fed
27.8±2.6 34.3±4.8 41.7 ± 8.1
88.9±2.2 72.2 ±5.1 82.2 ± 1.2**
37.5 ±3.1 32.7 ±3.8 30.9 ±2.2
8.7 ± 1.3 7.8±0.8 9.8± 1.1*
11.8 ± 1.1 12.1 ±0.6 8.4±0.8***
ders revealed essentially no difference in the sialic acid content (28-42 nmol/mg protein), but the fucose level of the membranes was significantly (p < 0.01) increased in the LP group compared to controls (table 4). These findings were also substantiated by the lec tin-binding data presented in table 4. The binding of WGA (affinity for Z)-GlcNAc and sialic acid) to brush borders from control and experimental pups was unaffected but a significant (p < 0.05) increase in the binding of UEAi (affinity for L-fucose) to the mem branes from the LP group was noticed. PNA specifically recognizes (3-Z>-Gal( 1— 3)-D-GalNAc in glycopeptides [19]. The binding of PNA to the disaccharide is gener
ally obliterated by sialic acid and fucose sub stitutions [6, 19]. Thus the reactivity of the membranes to PNA can also yield informa tion about membrane sialylation and fucosylation processes. The binding of PNA to brush borders in the LP-fed group was re duced (p < 0.001) compared to the control group. Analysis o f(14C)-D-mannose and (14C)-DGlcNAc incorporation into MVM showed no significant change in mannose incorpora tion in control and experimental animals. But the incorporation of labelled GlcNAc into membranes was reduced (24%) in pups nursed on rats fed 8% protein diet (table 5). Essentially similar results on the incorpora-
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Values are mean ± SD of 4-6 observations. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to pair-fed controls. 1 nmol/mg protein. 2 pg l25I-lectin bound/mg protein.
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Maternal Nutrition and Intestinal Glycosylation
TableS. (U-l4C)-D-mannose and (U-l4C)-GlcNAc incorporation into MVM of suckling pups nursed on mothers fed LP diet Group
Ad-libitum-fed control Pair-fed control LP fed
(U-l4C)-D-mannose
(U-l4C)-GlcNAc
native membrane1
delipidated membrane1
native membrane1
delipidated membrane1
2.39 ±0.47 1.18 ± 0.08 1.35 ±0.14
1.39 ±0.07 1.05 ±0.25 1.24 ±0.11
21.98 ± 3.31 20.48 ± 1.56 15.72 ± 2.61*
13.27 ±0.75 11.94 ±0.64 9.21 ±0.06**
tion of labelled mannose and GlcNAc were obtained with delipidated membranes under these conditions.
Discussion The results presented in this paper dem onstrate that weanling rats nursed on moth ers fed LP diet exhibit a considerable in crease in the absorption of 125I-labelled pro teins from intestine. The absorption of pro teins was determined both by TCA precipi tation and F.I.ISA, which yielded qualita tively identical results, but there were quan titative differences in the absorption data under these conditions [11], The observed increase in macromolecule absorption was associated with the enhanced binding of the proteins to the epithelial cell surface under these conditions. Intestinal tissue in the peri natal period is characterized by high-pinocytotic activity resulting in the absorption of macromolecules [18]. Since the imposition of undernutrition during the early suckling
period delays the maturational development of intestinal functions [20], such a phenome non may explain the observed increase in the absorption of BSA, y-globulin and a-lactalbumin in pups reared on lactating rats given LP diet. This assertion is corroborated by the observed decrease in the body and intes tinal weight of suckling rats in the LP group compared to controls. The present observa tions also indicate that suckling rats from the LP group had a greater absorption of macro molecules till day 17 after birth, a develop mental period when the pinocytotic activity of the intestinal epithelium commences to decline [18], Intestinal glycosylation, in particular the fucosylation, of microvillus surface is also affected in weanling rats nursed on mothers given LP diet. The membrane fucose levels and the binding of a-L-fucose-specific lectin, UEAi, were considerably more in pups from LP-diet-fed lactating rats. Interestingly, Biol et al. [7] have shown that feeding a semisyn thetic diet to adult rats affects the fucosyltransferase levels without altering the sialyl-
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25 pCi/100 g body weight of the labelled sugar was injected intraperitoneally. Animals were sacrificed after 2.5 h. Membranes were delipidated by treatment with chloroform:mcthanol (2:1, v/v) as described [5]. Values are mean ± SD of 4-6 observations. * p < 0.01; ** p < 0.001 compared to pair-fed controls. 1 cpm/mg protein X 10~3.
transferase activity in intestine. This sug gests the sensitivity of the intestinal fucosylation process to variations in the animal diet. Our results on the chemical analysis of sialic acid and fucose- and lectin-binding characteristics of brush borders in suckling animals from the LP group are in agreement to the observations in adult animals [7], Al though, at present, no information is avail able on glycosyl-transferase activities of in testinal tissue in weanling rats subjected to nutritional stress, it is likely that the ob served changes in the fucosylation pattern of microvillus surface may be related to altera tions in fucosyl-transferase levels under these conditions. However, changes in the availability of substrates, fucosyl-substitution-reactive sites and various cofactors in volved in the fucosylation may also be impli cated in the observed aberrations of epithe lial cell surface glycosylation in response to maternal nutrition. The present observa tions that feeding LP diet to lactating rats results in the augmentation of macromolecular transport as well as in the fucosylation of microvillus surface with no change in its sialylation may not be solely related to a ‘de layed’ maturational developmental phenom enon, as the stimulation of epithelial cell sur face fucosylation and cessation of macromolecular transport are characteristic determi nants of intestinal maturation [6, 18]. This may reflect that ontogeny of these two pro cesses is controlled by different mechanisms in rat intestine. We have previously shown that PNAreactive residues on MVM in the perinatal period are covered by sialic acid and by fucose substitution in the weaned animals [6], The present findings indicate that feed ing LP diet to lactating rats does not affect the sialic acid level but enhances the fucose
Babbar/Jaswal/Gupta/Mahmood
content of brush borders in weanling rats. Thus, the observed decrease in PNA-binding to brush borders in the LP-diet-fed group is a consequence of enhanced membrane fucosy lation rather than its effect on membrane sialylation. Incorporation of (14C)-Z)-mannose and ( i4C)-G1cNAc into MVM indicated a signifi cant decrease in GlcNAc incorporation with no change in mannose incorporation in pups nursed on rats fed LP diet. Thus the intesti nal mannosylation and N-acetylglucosamination processes are differently affected un der these conditions. Essentially similar re sults on the incorporation of labelled sugars were obtained in delipidated MVM. This also indicated that the observed glycosyla tion changes are confined to membrane glycopeptides which contained over 60% of the radioactivity in the native membranes. Alterations in glycosylation pattern of mi crovillus surface in weanling rats in response to maternal nutrition may influence the sen sitivity of intestinal epithelium to trophic factors present in the milk [21], This would obviously affect the maturational develop ment of intestine. The observed modifica tions in brush-border glycosylation in re sponse to maternal nutrition may also in fluence the microorganism-enterocyte inter action and hence the intestinal microflora. Whether the observed changes in membrane glycosylation of enterocytes under these con ditions are related to the development of various brush-border enzymes remains to be elucidated. Acknowledgement Financial assistance from the Indian Council of Medical Research, New Delhi, through project No. 8604320 is gratefully acknowledged.
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1 Montreuil J: Primary structure of glycoprotein glycans. Basis for the molecular biology of glyco proteins. Adv Carbohydr Chcm Biochem 1980; 37:157-227. 2 Talmadge KW, Burger MM: Carbohydrates and cell surface phenomenon. Int Rev Sci [Biochem] 1978;3:43-93. 3 Feizi T, Childs RA: Carbohydrates as antigenic determinants of glycoproteins. Biochem J 1987; 245:1-11. 4 Kenny AJ, Booth AJ: Microvilli: Their ultrastruc ture, enzymology and molecular organization; in Campbel PN, Aldrige WH (eds): Essays in Bio chemistry. New York, Acad Press, 1978, pp 1-44. 5 Mahmood A, Torres-Pinedo R: Postnatal changes in lectin binding to microvillus membrane from rat intestine. Biochem Biophys Res Commun 1983;113:400-406. 6 Torres-Pinedo R, Mahmood A: Postnatal changes in biosynthesis of microvillus membrane glycans of rat small intestine: I. Evidence of a develop mental shift from terminal sialylation to fucosylation. Biochem Biophys Res Commun 1984; 125: 546-553. 7 Biol MC, Martin A, Paulin C, et al: Glycosyltransferase activities in the rat intestinal mucosa: Comparison between standard commercial and semi-synthetic diets. Ann Nutr Metab 1984;28: 52-64. 8 Biol MC, Martin A, Richard M, Louisot P: Devel opmental changes in intestinal glycosyl-transferase activities. Pediatr Res 1987;22:250-256. 9 Mellies MJ, Ishikowa TT, Garlside PS, et al: Effects of varying maternal dietary fatty acids in lactating women and their infants. Am J Clin Nutr 1979;32:299-303. 10 Harzer G, Dieterich I, Haug M: Effects of the diet on the composition of human milk. Ann Nutr Metab 1984;28:231-239. 11 Udall JN, Pang K, Fritze L, et al: Development of gastrointestinal barrier. I. The effect of age on intestinal permeability to macromolecules. Pe diatr Res 1981;15:241-244. 12 McLean E and Ash R: The time course of appear ance and net accumulation of horse-radish perox idase (HRP) presented orally to juvenile Carp cyprinuscarpio(L). Comp Biochem Physiol 1986; 84A:687-690.
13 Kessler M, Acuto O, Storelli C, et al: A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating some properties of D-glucose and choline trans port systems. Biochim Biophys Acta 1978;506: 136-154. 14 Babba: HS, Jaswal VMS, Mahmood A: Effect of surface desialylation on intestinal uptake of pro teins in suckling rats. Indian J Exp Biol 1988;26: 31-33. 15 Dische Z, Shettles LB: A specific colour reaction of methylpentoses and a spectrophotometric mi cromethod for their determination. J Biol Chem 1948;175:595-603. 16 Skoza L, Mohos S: Stable thiobarbituric acid chromophore with dimethyl sulphoxide. Applica tion to sialic acid assay in analytical de-O-acetylation. Biochem J 1976;159:457-462. 17 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol re agent. J Biol Chem 1951;193:265-275. 18 Daniels VG, Hardy RN, Malinowksa KW: The effect of adrenalectomy or pharmacological inhi bition of adrenocortical function on macromoleculc uptake by the newborn rat intestine. J Physiol 1973;229:697-707. 19 Lotan R, Skutelsky E, Danon D, Sharon N: The purification composition and specificity of the anti-Tlectin from peanut (Arachis hypogea). J Biol Chem 1975;250:8518-8523. 20 Pathak RM, Mahmood A, Dudeja PK, Subrahmanyam D: Intestinal brush border membrane struc ture and function: Effect of early postnatal under nutrition. Pediatr Res 1981;15:112-114. 21 Sheard NF, Walker WA: The role of breast milk in the development of the gastrointestinal tract. Nutr Rev 1988;46:1-8.
Dr. Akhtar Mahmood Department of Biochemistry Panjab University Chandigarh 160 014 (India)
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References
Biol Neonate 1990;58:104-111
Intestinal Absorption of Macromolecules and Epithelial Cell Surface Glycosylation in Suckling Rats Nursed on Mothers Fed Low-Protein Diet-I HarjitS. Babbar, Virender M.S. Jaswal, Reena Gupta, Akhtar Mahmood Department of Biochemistry, Panjab University, Chandigarh, India
Key Words. Maternal nutrition • Macromolecular absorption • Membrane glycosylation • Lectin binding • Sialylation • Fucosylation
Introduction Surface oligosaccharides play a vital role in various cell functions; these include cell differentiation and recognition. They act as receptors for hormones, toxins and lectins and are involved in membrane transport processes [1-3]. The microvillus membrane (MVM) lining the enterocytes contains con
siderable amounts of carbohydrates [4], We have previously shown that surface-reactive carbohydrates, in particular sialic acid and fucose, undergo striking modifications dur ing postnatal development in rat intestine [5,6]. During early suckling period, the membrane is rich in sialic acid and contains low levels of fucose but the pattern is re versed on weaning [6], Biol et al. [7, 8] have
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Abstract. The effect of feeding 8 % protein (low-protein; LP) diet to lactating rats has been studied on the absorption of macromolecules and the glycosylation of enterocytes in suckling animals. The absorption of 125I-labelled bovine serum albumin, y-globulin and a-lactalbumin was 24-314 % more in pups nursed on mothers fed LP diet compared to the pair-fed controls. The observed enhancement in protein absorption was associated with a significant increase in the binding of these proteins to microvillus membranes (MVM). The sialic acid content of brush borders was unaltered but the fucose level was augmented (p < 0.01) in pups nursed on rats fed LP diet. The binding of 125I-labelled wheat germ agglutinin and Ulex europeus agglutinin I to MVM was in agreement to the data on sialic acid and fucose levels of the membranes. The binding of peanut agglutinin to MVM was 30% low in pups from LPdiet-fed animals. The incorporation of (l4C) Z)-mannose into MVM was essentially unaf fected but that of (l4C) N-acetylglucosamine was reduced in pups reared on mothers given LP diet. These findings suggest that the quality of maternal nutrition affects the absorption of macromolecules and the glycosylation in developing rat intestine.
105
Maternal Nutrition and Intestinal Glycosylation
Materials and Methods Albino rats (Wistar strain) were used. Pregnant animals were fed on a stock diet (Hindustan Lever, Bombay, India) with free access to water. On day 1, postparturition, lactating rats were fed on LP diet (ta ble 1) for 16 days. Control animals were pair-fed. Ani mals in the normal-fed group were given a rat pellet diet (18% protein) ad libitum. There were 6-8 pups per mother and at least 4 litters of each group were employed. On day 17 after birth, overnight-fasted pups were sacrificed under light ether anesthesia. Starting from the ligament of Treitz, entire intestine was removed, thoroughly washed in ice-cold saline and used for various biochemical studies. Absorption o f 125¡-Labelled Proteins The absorption of 125I-bovine serum albumin (BSA), y-globulin and a-lactalbumin was studied in vivo [11]. 0.2 ml of phosphate-buffered saline (PBS), pH 7.2 containing 300-500 pg BSA or y-globulin or a-lactalbumin and trace amounts of l2SI-labelled pro teins (specific activity 3 X 104—5 X 105 cpm/pg pro tein) were administered orally to each rat (body weight 18-22 g) using Ryle’s tube. Animals were sac rificed after 2 h. Blood was collected directly from the heart in heparinized tubes. The intestinal tissue was washed with PBS, blotted, weighed and the radioac tivity was determined in y-counter. For measuring the absorption of proteins into blood, suitable volume of the blood was treated with trichloroacetic acid (TCA) to a final concentration of 10% and the tubes were left
Table 1. The composition of control and LP diets fed to lactating rats Ingredient
Control, g
LP, g
Casein Starch/sucrose Cellulose Com oil Salt mixture Vitamin mixture
18 50 14 10 6 2
8 63 11 10 6 2
100 g of each diet provides 380 calories.
overnight at 4 °C. The contents were centrifuged and the supernatant was removed. Precipitates were washed twice with 10% TCA and the radioactivity was determined. The absorption of proteins was ex pressed as pg of l25I-protein/ml of blood. Measurement o f Protein Absorption by ELISA Antibodies against BSA and y-globulin were raised in rabbits by injecting 1.5 mg i.m. of the pro tein along with 250 pi of Freund's complete adjuvant. After 3 weeks, the same amount of the protein mixed with incomplete adjuvant was injected into each ani mal, followed by two more injections with a gap of 15 days each. Blood was obtained by cardiac puncture, allowed to clot at room temperature and was centri fuged at 3,000 g for 10 min. Blood serum was re moved and stored at -2 0 ° C . The presence of anti bodies against BSA and y-globulin was detected by immunodiffusion, carried out in agarose plates. Immunoreactive BSA and y-globulin in the serum of pups from the control and LP groups was detected by ELISA, using radish-peroxidase-labelled antirabbit antibodies as described by McLean and Ash [ 12]. The absorption of proteins into blood was expressed as pg/ml blood. Preparation o f MVM Brush borders were isolated and purified follow ing the method of Kessler et al. [13]. The membranes, suspended in 50 mM sodium maleate buffer (pH 6.8), exhibited a 12- to 15-fold enrichment of brush-border sucrase and alkaline phosphatase activities.
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described that the quality as well as the quantity of diet affects the glycosyl-transferase levels in intestine. Since milk is the sole source of nutrition for the developing pups during the suckling period and the composi tion of milk is altered in response to changes in the maternal nutrition [9, 10], we studied the effect of feeding low-protein (LP) diet (8% protein) to lactating rats on the absorp tion of 125I-labelled proteins and the glycosy lation of intestinal microvillus membrane in suckling rats.
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106
Fig. 1. Growth curve of suckling rats: control (A); pair-fed control (•) and LP groups (■). Bars indicate SD of the means.
immediately through 0.2 pm Millipore filter (EGWP, Milliporc Corporation. Bedford, Tex., USA) under suction. The filters were washed twice with 3 ml of PBS and the radioactivity retained on the filter was counted with a y-counter. Suitable controls to deter mine nonspecific protein binding were run simulta neously [6]. After correcting for the nonspecific bind ing, the protein/lectin binding to the membranes was expressed as pg of protein/lectin-bound/mg mem brane protein. Fucose content of the membranes was determined colorimetrically after initial hydrolysis of the membranes with 0.1 N H^SOj for 4 h at 100 °C [15]. Sialic acid was determined by the method of Skoza and Mohos [ 16] after digesting the membranes with 0.1 N H2SO4 for 60 min at 80 °C. Protein was determined according to Lowry et al. [17] using BSA as the standard. Statistical analysis of the data was done by Stu dent’s t test. All the reagents used were of analytical grade.
Labelling o f Proteins and Lectins with Various proteins and lectins were labelled with l25I as described earlier [14], 0.2 ml of reaction mix ture containing 100 pg protein or lectin, 0.3 mCi N al25I, 500 pg chloramine T and 0.2 M phosphate buffer (pH 7.2) was incubated at 30 °C. After 60 s. the reaction was terminated by adding 0.1 ml of sodium metabisulphite (lOmg/ml). Unreacted 125I was re moved by passing the mixture through Scphadex G 25 column (0.5 X 15 cm) followed by exhaustive dial ysis against 20 mA/Tris-HCl (pH 7.2). Specific activ ity of various labelled protcins/lcctins ranged between 2 X 104-5 X 10s cprn/pg protein. Binding o f¡ - L a b e lle d Proteins or Lectins to MVM The binding of BSA, y-globulin. a-lactalbumin and various lectins namely wheat germ agglutinin (WGA), Ulex europeus agglutinin (UEA[) and peanut agglutinin (PNA) to brush borders was measured as described previously [6]. In a total volume of 0.2 ml, 30 pg membrane protein was incubated with 2-4 pg of l25l-labelled protein or lectin in PBS (pH 7.2) at 30 °C for 30 min. Reaction was terminated by adding 4 ml of ice-cold PBS and the contents were filtered
As shown in figure 1, the gain in the body weight of pups weaned on dams given, LP diet was relatively small compared to that in the control group. At the time of sacrifice, the body weight of pups in the LP group was 35% low compared to controls. The intesti nal weight of suckling rats was also signifi cantly (p < 0.01) low in the LP group (0.47 ± 0.08 g) compared to control pups (0.82 ± 0.14 g). However, there was no difference in the intestinal length (39.2-41.4 cm) under these conditions. The intestinal tissue during the perinatal period exhibits high pinocytotic activity re sulting in the absorption of intact macromol ecules, and this process ceases on weaning [18], Thus, the absorption of l25I-BSA, yglobulin and a-lactalbumin was investigated in pups from control and experimental groups. The appearance of BSA in blood was 135% higher in the LP group compared to
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Results
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Table 2. Effect of feeding LP diet to lactating mothers on the absorption of labelled protein in suckling rats Group
BSA1
y-Globulin1
a-Lactalbumin1
Ad-libitum-fed control
TCA precipitate ELISA
1.33 ± 0.58 0.50 ±0.08
0.11 ±0.01 0.04 ±0.01
0.40 ±0.01
Pair-fed control
TCA precipitate ELISA
1.07 ±0.70 0.41 ±0.12
0.07±0.01 0.03 ±0.01
0.55 ±0.03
LP Fed
TCA precipitate ELISA
2.51 ±0.69* 0.92±0.10**
0.29 ±0.02** 0.16 ±0.01**
0.68 ±0.05**
the pair-fed controls (table 2). Nearly a 4fold increase in y-globulin and a significant (p < 0.001) elevation in a-lactalbumin ab sorption was observed under these condi tions. Measurement of BSA and y-globulin in the serum by ELISA also revealed a marked increase (p < 0.001) in the absorp tion of these proteins in pups nursed on lac tating rats fed LP diet. Still, the uptake val ues obtained by the TCA precipitation method were considerably higher compared to those obtained by ELISA. Such a differ ence in the detection of serum proteins by TCA precipitation and immunodiffusion methods has earlier been reported by Udall et al. [11]. Analysis of l25I-labelled proteins in intestinal tissue also revealed a significant increase in the protein uptake in pups from the LP group compared to controls (results not shown). This indicated that both the tis sue uptake of proteins and their absorption into blood was augmented in pups nursed on rats fed LP diet. In has been shown that in vitro uptake of BSA, y-globulin and a-lactalbumin follows a saturation phenomenon suggesting the in
volvement of a receptor-mediated process for their uptake [14]. Therefore, the binding of 125I-labelled proteins to MVM was also examined under these conditions. As shown in table 3, the binding of BSA, y-globulin and a-lactalbumin to MVM was 20-37% higher in the LP group compared to the pair-fed control. Thus, the observed increase in the absorption of proteins in suckling rats is pre sumably a consequence of enhanced binding of the macromolecules to the epithelial cell surface in response to LP diet given to the lactating rats. It may also be pointed out that there was essentially no difference in body weight, absorption of proteins, binding data and membrane glycosylation between ad li bitum controls and pair-fed groups. There fore, the results obtained from pups weaned on dams given LP diet were compared with those from the pair-fed animals, hence re ferred as ‘controls’. Sialic acid and fucose are interesting sug ars, which generally occupy the nonreducing terminal ends and thus act as stoppers of oligosaccharidic chains in glycopeptides [1,2], Analysis of these sugars in brush bor-
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Values are mean ± SD of 4-6 observations. * p < 0.05 and ** p < 0.001 compared to pair-fed controls. 1 pg protein absorbed/ml blood.
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Table 3. Binding of l25I-labelled proteins to microvillus membranes from suckling rats nursed on mothers fed LP diet Group
BSA1
y-Globulin1
a-Lactalbumin1
Ad-libitum-fed control Pair-fed control LP fed
2.4±0.2 1.9 ±0.4 3.1 ±0.1**
5.5 ±0.6 6.1 ±0.2 9.2± 1.5**
6.2±0.2 6.7±0.2 8.4±0.5*
Values are mean ± SD of 4-6 observations. * p < 0.05 and ** p < 0.01 compared to pair-fed controls. 1 pg protein bound/mg protein.
Table 4. The binding of l25l-labelled WGA, UEAi and PNA to MVM of suckling animals nursed on mothers fed LP diet Group
Sialic acid1
Fucose1
WGA2
UEAi2
PNA2
Ad-libitum-fed control Pair-fed control LP fed
27.8±2.6 34.3±4.8 41.7 ± 8.1
88.9±2.2 72.2 ±5.1 82.2 ± 1.2**
37.5 ±3.1 32.7 ±3.8 30.9 ±2.2
8.7 ± 1.3 7.8±0.8 9.8± 1.1*
11.8 ± 1.1 12.1 ±0.6 8.4±0.8***
ders revealed essentially no difference in the sialic acid content (28-42 nmol/mg protein), but the fucose level of the membranes was significantly (p < 0.01) increased in the LP group compared to controls (table 4). These findings were also substantiated by the lec tin-binding data presented in table 4. The binding of WGA (affinity for Z)-GlcNAc and sialic acid) to brush borders from control and experimental pups was unaffected but a significant (p < 0.05) increase in the binding of UEAi (affinity for L-fucose) to the mem branes from the LP group was noticed. PNA specifically recognizes (3-Z>-Gal( 1— 3)-D-GalNAc in glycopeptides [19]. The binding of PNA to the disaccharide is gener
ally obliterated by sialic acid and fucose sub stitutions [6, 19]. Thus the reactivity of the membranes to PNA can also yield informa tion about membrane sialylation and fucosylation processes. The binding of PNA to brush borders in the LP-fed group was re duced (p < 0.001) compared to the control group. Analysis o f(14C)-D-mannose and (14C)-DGlcNAc incorporation into MVM showed no significant change in mannose incorpora tion in control and experimental animals. But the incorporation of labelled GlcNAc into membranes was reduced (24%) in pups nursed on rats fed 8% protein diet (table 5). Essentially similar results on the incorpora-
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Values are mean ± SD of 4-6 observations. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to pair-fed controls. 1 nmol/mg protein. 2 pg l25I-lectin bound/mg protein.
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TableS. (U-l4C)-D-mannose and (U-l4C)-GlcNAc incorporation into MVM of suckling pups nursed on mothers fed LP diet Group
Ad-libitum-fed control Pair-fed control LP fed
(U-l4C)-D-mannose
(U-l4C)-GlcNAc
native membrane1
delipidated membrane1
native membrane1
delipidated membrane1
2.39 ±0.47 1.18 ± 0.08 1.35 ±0.14
1.39 ±0.07 1.05 ±0.25 1.24 ±0.11
21.98 ± 3.31 20.48 ± 1.56 15.72 ± 2.61*
13.27 ±0.75 11.94 ±0.64 9.21 ±0.06**
tion of labelled mannose and GlcNAc were obtained with delipidated membranes under these conditions.
Discussion The results presented in this paper dem onstrate that weanling rats nursed on moth ers fed LP diet exhibit a considerable in crease in the absorption of 125I-labelled pro teins from intestine. The absorption of pro teins was determined both by TCA precipi tation and F.I.ISA, which yielded qualita tively identical results, but there were quan titative differences in the absorption data under these conditions [11], The observed increase in macromolecule absorption was associated with the enhanced binding of the proteins to the epithelial cell surface under these conditions. Intestinal tissue in the peri natal period is characterized by high-pinocytotic activity resulting in the absorption of macromolecules [18]. Since the imposition of undernutrition during the early suckling
period delays the maturational development of intestinal functions [20], such a phenome non may explain the observed increase in the absorption of BSA, y-globulin and a-lactalbumin in pups reared on lactating rats given LP diet. This assertion is corroborated by the observed decrease in the body and intes tinal weight of suckling rats in the LP group compared to controls. The present observa tions also indicate that suckling rats from the LP group had a greater absorption of macro molecules till day 17 after birth, a develop mental period when the pinocytotic activity of the intestinal epithelium commences to decline [18], Intestinal glycosylation, in particular the fucosylation, of microvillus surface is also affected in weanling rats nursed on mothers given LP diet. The membrane fucose levels and the binding of a-L-fucose-specific lectin, UEAi, were considerably more in pups from LP-diet-fed lactating rats. Interestingly, Biol et al. [7] have shown that feeding a semisyn thetic diet to adult rats affects the fucosyltransferase levels without altering the sialyl-
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25 pCi/100 g body weight of the labelled sugar was injected intraperitoneally. Animals were sacrificed after 2.5 h. Membranes were delipidated by treatment with chloroform:mcthanol (2:1, v/v) as described [5]. Values are mean ± SD of 4-6 observations. * p < 0.01; ** p < 0.001 compared to pair-fed controls. 1 cpm/mg protein X 10~3.
transferase activity in intestine. This sug gests the sensitivity of the intestinal fucosylation process to variations in the animal diet. Our results on the chemical analysis of sialic acid and fucose- and lectin-binding characteristics of brush borders in suckling animals from the LP group are in agreement to the observations in adult animals [7], Al though, at present, no information is avail able on glycosyl-transferase activities of in testinal tissue in weanling rats subjected to nutritional stress, it is likely that the ob served changes in the fucosylation pattern of microvillus surface may be related to altera tions in fucosyl-transferase levels under these conditions. However, changes in the availability of substrates, fucosyl-substitution-reactive sites and various cofactors in volved in the fucosylation may also be impli cated in the observed aberrations of epithe lial cell surface glycosylation in response to maternal nutrition. The present observa tions that feeding LP diet to lactating rats results in the augmentation of macromolecular transport as well as in the fucosylation of microvillus surface with no change in its sialylation may not be solely related to a ‘de layed’ maturational developmental phenom enon, as the stimulation of epithelial cell sur face fucosylation and cessation of macromolecular transport are characteristic determi nants of intestinal maturation [6, 18]. This may reflect that ontogeny of these two pro cesses is controlled by different mechanisms in rat intestine. We have previously shown that PNAreactive residues on MVM in the perinatal period are covered by sialic acid and by fucose substitution in the weaned animals [6], The present findings indicate that feed ing LP diet to lactating rats does not affect the sialic acid level but enhances the fucose
Babbar/Jaswal/Gupta/Mahmood
content of brush borders in weanling rats. Thus, the observed decrease in PNA-binding to brush borders in the LP-diet-fed group is a consequence of enhanced membrane fucosy lation rather than its effect on membrane sialylation. Incorporation of (14C)-Z)-mannose and ( i4C)-G1cNAc into MVM indicated a signifi cant decrease in GlcNAc incorporation with no change in mannose incorporation in pups nursed on rats fed LP diet. Thus the intesti nal mannosylation and N-acetylglucosamination processes are differently affected un der these conditions. Essentially similar re sults on the incorporation of labelled sugars were obtained in delipidated MVM. This also indicated that the observed glycosyla tion changes are confined to membrane glycopeptides which contained over 60% of the radioactivity in the native membranes. Alterations in glycosylation pattern of mi crovillus surface in weanling rats in response to maternal nutrition may influence the sen sitivity of intestinal epithelium to trophic factors present in the milk [21], This would obviously affect the maturational develop ment of intestine. The observed modifica tions in brush-border glycosylation in re sponse to maternal nutrition may also in fluence the microorganism-enterocyte inter action and hence the intestinal microflora. Whether the observed changes in membrane glycosylation of enterocytes under these con ditions are related to the development of various brush-border enzymes remains to be elucidated. Acknowledgement Financial assistance from the Indian Council of Medical Research, New Delhi, through project No. 8604320 is gratefully acknowledged.
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1 Montreuil J: Primary structure of glycoprotein glycans. Basis for the molecular biology of glyco proteins. Adv Carbohydr Chcm Biochem 1980; 37:157-227. 2 Talmadge KW, Burger MM: Carbohydrates and cell surface phenomenon. Int Rev Sci [Biochem] 1978;3:43-93. 3 Feizi T, Childs RA: Carbohydrates as antigenic determinants of glycoproteins. Biochem J 1987; 245:1-11. 4 Kenny AJ, Booth AJ: Microvilli: Their ultrastruc ture, enzymology and molecular organization; in Campbel PN, Aldrige WH (eds): Essays in Bio chemistry. New York, Acad Press, 1978, pp 1-44. 5 Mahmood A, Torres-Pinedo R: Postnatal changes in lectin binding to microvillus membrane from rat intestine. Biochem Biophys Res Commun 1983;113:400-406. 6 Torres-Pinedo R, Mahmood A: Postnatal changes in biosynthesis of microvillus membrane glycans of rat small intestine: I. Evidence of a develop mental shift from terminal sialylation to fucosylation. Biochem Biophys Res Commun 1984; 125: 546-553. 7 Biol MC, Martin A, Paulin C, et al: Glycosyltransferase activities in the rat intestinal mucosa: Comparison between standard commercial and semi-synthetic diets. Ann Nutr Metab 1984;28: 52-64. 8 Biol MC, Martin A, Richard M, Louisot P: Devel opmental changes in intestinal glycosyl-transferase activities. Pediatr Res 1987;22:250-256. 9 Mellies MJ, Ishikowa TT, Garlside PS, et al: Effects of varying maternal dietary fatty acids in lactating women and their infants. Am J Clin Nutr 1979;32:299-303. 10 Harzer G, Dieterich I, Haug M: Effects of the diet on the composition of human milk. Ann Nutr Metab 1984;28:231-239. 11 Udall JN, Pang K, Fritze L, et al: Development of gastrointestinal barrier. I. The effect of age on intestinal permeability to macromolecules. Pe diatr Res 1981;15:241-244. 12 McLean E and Ash R: The time course of appear ance and net accumulation of horse-radish perox idase (HRP) presented orally to juvenile Carp cyprinuscarpio(L). Comp Biochem Physiol 1986; 84A:687-690.
13 Kessler M, Acuto O, Storelli C, et al: A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating some properties of D-glucose and choline trans port systems. Biochim Biophys Acta 1978;506: 136-154. 14 Babba: HS, Jaswal VMS, Mahmood A: Effect of surface desialylation on intestinal uptake of pro teins in suckling rats. Indian J Exp Biol 1988;26: 31-33. 15 Dische Z, Shettles LB: A specific colour reaction of methylpentoses and a spectrophotometric mi cromethod for their determination. J Biol Chem 1948;175:595-603. 16 Skoza L, Mohos S: Stable thiobarbituric acid chromophore with dimethyl sulphoxide. Applica tion to sialic acid assay in analytical de-O-acetylation. Biochem J 1976;159:457-462. 17 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol re agent. J Biol Chem 1951;193:265-275. 18 Daniels VG, Hardy RN, Malinowksa KW: The effect of adrenalectomy or pharmacological inhi bition of adrenocortical function on macromoleculc uptake by the newborn rat intestine. J Physiol 1973;229:697-707. 19 Lotan R, Skutelsky E, Danon D, Sharon N: The purification composition and specificity of the anti-Tlectin from peanut (Arachis hypogea). J Biol Chem 1975;250:8518-8523. 20 Pathak RM, Mahmood A, Dudeja PK, Subrahmanyam D: Intestinal brush border membrane struc ture and function: Effect of early postnatal under nutrition. Pediatr Res 1981;15:112-114. 21 Sheard NF, Walker WA: The role of breast milk in the development of the gastrointestinal tract. Nutr Rev 1988;46:1-8.
Dr. Akhtar Mahmood Department of Biochemistry Panjab University Chandigarh 160 014 (India)
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References
