BIOCHEMICAL
MEDICINE
AND
METABOLIC
BIOLOGY
43, 45-52 (1990)
Effect of Undernutrition and Hormone Treatments on the Absorption of Proteins in Suckling Rat Intestine HARJIT S. BABBAR,
VIRENDER M. S. JASWAL, AND AKHTAR MAHMOOD'
Department of Biochemistry,
Panjab University, Chandigarh
160 014, India
Received July 28, 1989, and in revised form October 2, 1989
The intestinal epithelium during the perinatal period is characterized by a high rate of pinocytotic activity leading to the absorption of macromolecules and this process ceases upon weaning in rats (1,2). Administration of cortisone and thyroxine to suckling animals has been shown to induce precocious closure of intestinal tissue and abolishes the absorption of macromolecules (3,4). It has been demonstrated earlier that undernutrition imposed during the early suckling period considerably affects the development of brush border enzymes and transport functions of intestinal epithelium as a consequence of “delayed maturation” (5,6). However, it is not known whether the imposition of nutritional deficiency during weanling also affects the absorption of macromolecules from intestine. Therefore, the present studies were undertaken to examine the effect of undernutrition on the absorption of various proteins in suckling rat intestine. In addition, the effect of cortisone, thyroxine, and insulin on the absorption of macromolecules was also investigated in nutritionally deprived animals. MATERIALS AND METHODS All reagents were of analytical grade. BSA and y-globulin were obtained from Sigma. 12’1-labeled Na (sp. act 13.2 mCi/pg) was from Bhabha Atomic Research Center, Trombay (Bombay). Wistar strain albino rats were used in these studies. Animals were fed ad libitum rat pellet diet (Lipton India Ltd) and had free access to water. Undernutrition was induced following the method of Winick and Noble (7). There were 5-7 pups per mother in the control group, while the litter size was increased to 16-18 pups in the undernourished (UN) group. At Day 17 after birth UN pups were administered cortisone (25 mg/lOO g body wt, im), thyroxine (25 hg/lOO g body wt, ip), or insulin (10 mU/g body wt, ip) daily for 4 days. The corresponding control animals (saline-treated UN group) were age-matched and received saline alone. On Day 21, the overnight-fasted pups were sacrificed under ’ To whom correspondence
should be addressed. 45 0885-4505/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reprcduclion in any form reserved.
46
BABBAR,
JASWAL,
AND
MAHMOOD
ether anesthesia. The abdomen was opened and intestine removed, thoroughly washed with ice-cold saline, weighed, and used for biochemical studies. Since there were no differences in various parameters studied in untreated and salinetreated UN animals, results relating to saline-treated UN animals are referred to in this paper. Labeling of proteins with lz5Z. Various proteins were labeled with ‘251 as described previously (8). Briefly, 0.2 ml of reaction mixture containing 100 pg protein, 0.3 mCi ‘*’ I-labeled Na, 500 pg chloramine T, and 0.2 M phosphate buffer, pH 7.2, was incubated at 30°C. After 60 set, the reaction was terminated by adding 0.1 ml of sodium metabisulfite (10 mg/ml). Unreacted ‘25I was removed by passing the mixture through a Sephadex G-25 column (0.5 x 15 cm) followed by exhaustive dialysis against 20 mM Tris-HCl, pH 7.2. Specific activity of the labeled proteins varied between 2 x lo4 and 5 x 10’ cpm/pg protein. Absorption of ‘2’Z-lubeled proteins. The absorption of ‘2sI-labeled bovine serum albumin (BSA) and y-globulin was studied in viwo by the method of Udall et al. (9). Phosphate-buffered saline (PBS) (2 ml) pH 7.2, containing 400-500 pg BSA and y-globulin and trace amounts of 12?-labeled proteins was administered orally to each animal using a Ryle’s tube (25 ,ug/g body wt). Animals were sacrificed after 90 min. Blood was collected directly from the heart in heparinized tubes. A suitable volume of the blood was treated with trichloroacetic acid to a final concentration of 10% and the tubes were left overnight at 4°C. The tubes were centrifuged at 3000g for 5 min and the supematant was discarded. The precipitates were washed twice with 10% trichloroacetic acid and the radioactivity was directly determined in a gamma counter. Measurement of serum BSA and y-globulin by ELZSA. Antibodies against BSA and y-globulin were raised in rabbits (New Zealand White by injecting 1.5 mg of the protein (im) along with 250 ~1 of Freund’s complete adjuvant. After 3 weeks, same amount of the protein mixed with incomplete adjuvant was injected into each animal, followed by two more injections at 15-day intervals. Blood, obtained by cardiac puncture, was allowed to clot at room temperature and centrifuged at 3000g for 10 min. Blood sera were removed and stored at - 20°C. The presence of antibodies against BSA and y-globulin was detected by immunodiffusion carried out on agarose plates. Immunoreactive BSA and y-globulin in the serum was detected by ELISA using horseradish peroxidase-labeled anti-rabbit antibodies as described by McLeanI&d Ash (10). The absorption of various proteins into blood was expressed as micrograms protein per milliliter of blood. Isolation of microvilfus membranes (MVM). The method of Kessler et al. (11) was followed to prepare MVM. The membranes were suspended in 50 mM sodium maleate buffer (pH 6.8). They exhibited lo- to 13-fold enrichment of alkaline phosphatase and brush border lactase/sucrase activities compared to the tissue homogenates. Alkaline phosphatase was assayed by the method of Bergmeyer (12) and disaccharidases were determined by the method of Dahlqvist (13). Leucine amino peptidase activity was determined by the method described by Goldbarg and
MACROMOLECULARABSORF'TIONANDMALNUTRlTION
47
Rutenberg (14). Protein was measured according to Lowry et al. (15) using bovine serum albumin as the standard. Binding of ‘25Z-labeled proteins to MVM. The binding of labeled BSA and yglobulin to brush borders was determined by the method reported previously (8). Membranes (25-30 pg protein) were incubated with 2-4 pg 12?-labeled protein in PBS, pH 7.2, at 30°C for 30 min in a reaction volume of 0.2 ml. The reaction was stopped by adding 3 ml of PBS and filtering through a 0.2~pm Millipore filter (EGWP Millipore Corp., Bedford, MA) under suction. The filters were washed twice with 6 ml of ice-cold PBS and the radioactivity retained on the filters was determined in a gamma counter. Nonspecific protein binding to filters under these conditions amounted to 18% of the total radioactivity added to the assay mixture. After correction was made for the nonspecific binding, protein binding to MVM was calculated and expressed as picograms “‘I-labeled protein bound per milligram protein. Luminal degradation of ‘2sZ-labeled proteins. Labeled BSA and y-globulin (500 pg) was incubated with luminal fluid (obtained by flushing the intestine with 1 ml ice-cold saline) in a total volume of 1 ml at 37°C for 60 min. The reaction was stopped by placing the tubes in a boiling water bath for 3 min. The undegraded proteins were determined by ELISA (10). Statistical analysis of the data was done by Student’s t test. RESULTS
On Day 21 after birth, nutritionally deprived animals exhibited a considerable decrease in the body weight (35%) and intestinal weight (73%) compared to control animals (Table 1). There was essentially no difference in intestinal length (48-57 cm) under these conditions, The data on the effect of UN and hormone treatments on intestinal weight and length are also presented in Table 1. A 38% lower sucrase activity and a threefold higher lactase activity was observed in the brush borders of UN animals (Table 2). Alkaline phosphatase ac\tivity was also reduced by 26% in nutritionally deficient rats. Cortisone treatment of UN animals caused a significant (P < 0.001) stimulation of the sucrase activity and reduction (P < 0.001) of lactase activity in intestine. Similar results were obtained after thyroxine administration; however, insulin treatment reduced (P < 0.001) sucrase and lactase activities in UN animals. Leucine aminopeptidase activity remained unaltered in malnourished pups. Cortisone and insulin depressed leucine aminopeptidase activity by 25 and 44%, respectively, whereas thyroxine administration stimulated (56%) the enzyme activity in UN pups. The effect of UN on the absorption of ‘,251-labeled proteins was investigated in vivo. The appearance of BSA and y-globulin in the blood serum detected both by TCA precipitation and ELISA revealed a marked enhancement (P < 0.001) in UN animals compared to controls (Table 3). There was a considerable difference in the amount of labeled proteins detected in the sera using these two methods under various experimental conditions. The TCA precipitation method always yielded much higher values for ‘251-labeled BSA and y-globulin in blood as compared to those estimated by ELISA. There were also qualitative differences in the uptake of BSA and y-globulin estimated by these two methods.
17 16 11 10 12
Control UN UN + cortisone UN + thyroxine UN + insulin
23.1 15.1 13.5 14.2 13.9
+ 2.3 ,e_ 1.3* + 0.02 + 1.2 + 0.4
2.30 0.61 0.51 1.25 1.06
+ + + f +
0.41 0.05* 0.01” 0.03 o.22b
56.5 48.1 45.1 47.3 46.3
0.76 0.47 0.54 0.56 0.41
k k 2 k +:
0.05 0.02* 0.02” 0.01” 0.01”
Sucrase (units/mg 0.07 k 0.23 + 0.08 f 0.15 2 0.12 f
protein) 0.01 O.Ol* 0.001” 0.01” 0.01”
Lactase
1.20 0.89 0.79 1.21 0.99
+ + -t 2 f
0.07 0.04* O.O1lb 0.06 ow
Alkaline phosphatase
TABLE 2 and Hormone Treatments on Brush Border Enzymes in Suckling Rat Intestine
2.9 5.5 3.5 1.1 1.2
f k + + k
1.7 6.8* 6.4 3.2’ 9.1’
0.17 0.16 0.12 0.25 0.09
k k 2 + f
0.04 0.01 0.02” 0.01” 0.02”
Leucine amino peptidase
24.6 78.9 88.4 37.8 43.7
Intestinal length/ intestinal weight (cm/g)
Note. Values are means f SD of three to four preparations. One enzyme unit is equal to 1 pmole of the substrate transformed into product per minute under standard assay conditions. * P < 0.081 compared to control group. “P < 0.001; bP < 0.01; ‘P < 0.05 compared to UN group.
Control UN UN + cortisone UN + thyroxine UN + insulin
Group
Effect of Undernutrition
f + + ” +
Intestinal length (cm)
Note. Values are means f SD. n, number of animals. * P < 0.001 compared to control group. “P < 0.05; ‘P < 0.01; ‘P < 0.001 compared to UN group.
n
Group
Intestinal wt. (g)
TABLE 1 and Hormone Treatments on Certain Parameters in Suckling Rats
Body wt. (g)
Effect of Undernutrition
1.33 2.78 1.64 1.29 1.14
2 k f 2 f
0.58 0.37** 0.24” 0.12” 0.02”
TCA precipitation
BSA
0.131 0.763 0.077 0.060 0.053
2 k f k *
0.016 0.058* 0.006 0.004 0.004”
ELISA 1.21 1.80 1.10 1.11 0.80
* f ” + + blood.
0.10 0.22** 0.20” 0.10 0.18
y-Globulin
Suckling Rat Intestine
TCA precipitation
3 and Hormone-Injected
Note. Values are means -C SD of six to eight observations as micrograms protein absorbed per milliliter * P < 0.001; **P < 0.01 compared to control group. “P < 0.001 compared to UN group.
Control UN UN + cortisone UN + thyroxine UN + insulin
Group
Absorption
TABLE of ‘251-labeled Proteins in Undernourished
0.406 0.410 0.248 0.250 0.180
k 2 e + k
0.008 0.030* 0.002” 0.002” 0.001”
ELISA
E
z 2 2 ?
5
i z
$ $
G 0 s %
8
50
BABBAR,
JASWAL,
AND MAHMOOD
BSA uptake estimated by TCA precipitation was higher compared to the absorption of y-globulin; however, the opposite was the case for the uptake estimated by ELISA in UN pups (Table 3). Administration of cortisone, thyroxine, or insulin to UN pups significantly (P < 0.001) reduced the absorption of BSA and y-globulin compared to untreated UN pups. To examine the possibility whether the observed increase in the absorption of proteins is related to their binding to epithelial cell surface in malnourished animals, we studied the binding of ‘251-labeled BSA and y-globulin to MVM from control and experimental animals. These results are presented in Table 4. There was no significant difference in the binding of BSA and y-globulin to brush borders from control and UN pups. Cortisone and insulin treatments of UN animals also did not affect the binding of these proteins to the membranes. The proteolytic activity of luminal content was significantly higher in control animals compared to that in the UN pups: Control BSA = 303.1 -+ 24.9, yglobulin = 309.8 ? 12.8 versus 170.1 ? 4.2 and 199.5 + 3.1 pg/60 min/mg protein in UN pups, respectively. DISCUSSION The imposition of undernutrition in weanling rats resulted in considerable changes in the activities of various brush border enzymes. It is well established that lactase activity is high and sucrase activity is practically absent during the perinatal period. However, lactase activity begins to decline and sucrase activity increases around Day 14. Adult levels of these enzymes are reached by Day 21 after birth in rat intestine (1,16). The data presented indicate that nutritional restrictions imposed in suckling rats augmented lactase and diminished the suerase activities. This suggests that maturational development of these enzymes is delayed under these conditions (5,6). Administration of cortisone and thyroxine induced adult-type changes in various brush border enzymes in UN animals. These effects are essentially similar to their well-characterized effects in normal tissues (1,16). Insulin treatment of UN animals depressed both sucrase and lactase activities. These observations are not at par with the stimulatory action of insulin on these enzymes in suckling mice (17) and adult rat intestine (18). This may be due to species differences or it may reflect differences in the mode of action of insulin in affecting the development of brush border disaccharidases in normal and malnourished animals. Effect of Undernutrition
Group Control UN UN + cortisone UN + insulin
TABLE 4 and Hormones on the Binding of ‘251-labeled Proteins to Intestinal Microvillus Membrane in Suckling Rats BSA (pg protein bound/mg protein) 106.71 74.41 83.03 85.8
* I1 + 4.9 * 6.32 f 7.24
Note. Values are means 2 SD of three to five observations.
y-Globulin (pg protein bound/mg protein) 84.70 92.7 99.37 82.46
” 6.27 + 2.46 f 8.23 * 11.66
MACROMOLECULAR
ABSORPTION
AND
MALNUTRITION
51
UN pups exhibited a considerably higher absorption of 1251-labeled BSA and y-globulin compared to the controls. Since pinocytotic activity of the neonatal intestine is high (1,2), the observed increase in protein absorption in nutritionally deprived intestine may be attributed to the delayed maturational phenomenon. Worthington et al. (19) have also reported enhanced capacity of intestine to absorb intact proteins in protein-deficient animals. A similar increase in the absorption of BSA in malnourished rats has been described by Rothman et al. (20). Thus it appears that intestinal tissue responds to protein or calorie deficiency by augmenting the absorption of macromolecules. Administration of cortisone, thyroxine, and insulin in nutritionally deprived pups reduced the absorption of proteins almost to control levels. Administration of large doses of cortisone to 5-day-old pups is known to induce precocious closure of intestinal tissue, thus abolishing the absorption of macromolecules (3). Similar results have also been reported in hypophysectomized rats (4). The present findings indicate a common mode of action of cortisone, thyroxine, and insulin in depressing the absorption of proteins in UN animals. There was no difference in the binding of labeled BSA and y-globulin to brush borders from UN and hormone-injected UN pups compared to the controls. Therefore, the observed increase in the absorption of BSA and y-globulin in malnourished pups is unrelated to their binding to microvillus surface and is presumably a consequence of delayed maturational development, as revealed by disaccharidase patterns under these conditions. However, Israel et al. (21) have recently described a marked decrease in the absorption of IgG in thyroxineinjected suckling rats, which was related to the disappearance of IgG receptors on the epithelial cell surface. The observed increase in the absorption of various proteins in malnourished animals can also be attributed to reduced luminal degradation, as revealed by the present data. Telemo et al. (22) have also reported enhanced intraluminal proteolytic activity in weaned animals compared to that in the weanling rat intestine. The present findings evinced that 21-day-old rats exposed to malnutrition exhibited considerable transport of macromolecules. A number of diseased conditions, such as coeliac disease (23) viral and bacterial gastroenteritis, chronic alcoholism, and certain food allergies, are known to stimulate the transport of macromolecules from intestine (24). In conclusion, the results presented herein indicate that undernutrition imposed during suckling period enhances the absorption of macromolecules in rat intestine. The process is sensitive to cortisone, thyroxine, and insulin, suggesting the involvement of these hormones in the maturational development of the macromolecular transport process in intestine. SUMMARY The absorption of ‘251-labeled BSA and y-globulin was significantly (P < 0.01) elevated in UN pups compared to the controls. Administration of pharmacological doses of cortisone, thyroxine, and insulin markedly (P < 0.001) reduced the absorption of BSA and y-globulin in UN pups. There was no significant difference in the binding of ‘251-labeled BSA and y-globulin to microvillus membrane in the control and experimental animals. However, the degradation of labeled BSA and
52
BABBAR,
JASWAL,
AND MAHMOOD
y-globulin by luminal content was considerably higher (S-70%) in controls compared to UN pups. This suggested that observed increase in the absorption of proteins in nutritionally deprived pups was unrelated to their binding to the microvillus surface but presumably it is a consequence of reduced luminal degradation together with delayed maturational development as suggested by the pattern of brush border enzymes in the UN intestinal tissue. ACKNOWLEDGMENT These studies were supported by the Indian Council of Medical Research, New Delhi.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
Henning, S. J., and Kretchmer, N., Enzyme 15, 3 (1973). Udall, J. N., and Walker, W. A., J. Pediatr. Gastroenterol. Nutr. 1, 295 (1982). Jones, R. E., Biochim. Biophys. Acta 274, 412 (1972). Moog, F., and Yeh, K. Y., Dev. Biol. 69, 159 (1979). Pathak, R. M., Dudeja, P. K., Ansari, S., and Mahmood, A., Ann. Nutr. Metab. 26, 331 (1982). Pathak, R. M., Mahmood, A., Dudeja, P. K., and Subrahmanyam, D., Pediatr. Res. 15, 112 (1981). Winick, M., and Noble, A., J. Nutr. 89, 300 (1966). Babbar, H. S., Jaswal, V. M. S., and Mahmood, A., Indian J. Exp. Biol. 26, 31 (1988). Udall, J. N., Pang, K., Fritze, R. L., Kleinman, R., and Walker, W. A., Pediatr. Res. 15, 241 (1981). McLean, E., and Ash, R., Camp. Biochem. Physiol. A. 84, 687 (1986). Kessler, M., Acute, O., Storelli, C., Murer, H., Muller, M., and Semenza, G., Biochim. Biophys. Acta 506, 136 (1978). Bergmeyer, H. U., “Methods of Enzymatic Analysis,” p. 783. Academic Press, New York, 1%3. Dahlqvist, A., Anal. Biochem. 7, 18 (1964). Goldbarg, J. A., and Rutenberg, A. M., Cancer 11, 283 (1958). Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193, 265 (1951). Henning, S. J., Amer. J. Physiol. 241, Cl99 (1981). Menard, D., Malo, C., and Calvert, R., Dev. Biol. 85, 150 (1981). Mahmood, A., Pathak, R. M., and Agarwal, N., Experientia 34, 741 (1978). Worthington, B. S., Boatman, E. S., and Kenney, G. E., Amer. J. Clin. Nutr. 27, 276 (1974). Rothman, D., Lathan, M. C., and Walker, W. A., Nutr. Res. 2, 467 (1982). Israel, E. J., Pang, K. Y., Harmatz, P. R., and Walker, W. A., Amer. J. Physiol. 252, G762 (1987). Telemo, E., Westrom, B. R., Ekstram, G., and Karlssan, B. W., Biol. Neonate 52, 141 (1987). Cobden, I., Rothwell, J., and Axon, A. T. R., Gut 21, 512 (1980). Gardnar, M. L. G., Annu. Rev. Nutr. 8, 329 (1988).