The Effect of Diet on Lactase Activity in the Rat

Vol. 60, No.3 Printed in U. S. A.

GASTROENTEROLOGY

Copyright

1971 by The Williams & Wilkins Co.

THE EFFECT OF DIET ON LACTASE ACTIVITY IN THE RAT T. D. BOLIN, M.B., M.R.C.P., ANNE McKERN, AND A. E. DAVIS, M.D., M.R.C.P. School of Medicine, The University of New South Wales, Prince Henry Hospital, Sydney, Australia

A study was undertaken to adapt intestinal lactase in weanling rats and to correlate changes in lactase activity with in vivo lactose hydrolysis. The postweaning decline in lactase activity was not prevented by feeding a 10% lactose diet. Lactase activity subsequently rose at 8 weeks of age and remained high for the duration of lactose feeding. A decline in lactase activity occurred after withdrawal of lactose from the diet, and when lactose was reintroduced lactase activity rose. The time taken for these adaptive responses varied between 5 and 10 weeks. Changes in jejunal lactase activity were accompanied by similar alterations in both sucrase and maltase activities, while there was no alteration in another brush border enzyme, alkaline phosphatase. The increase in lactase with a pH optimum of 5.8, with slight alteration of the enzyme with a more acid pH optimum, was taken to be evidence for a brush border location. This was supported by the correlation between increased lactase activity and increased in vivo hydrolysis of 14C-1-lactose in isolated loops of intestine. Intestinal lactase activity is high in the suckling rat, and a postweaning decline in activity has been well documented. 1-6 This study was undertaken to determine the effect of prolonged lactose feeding. Was the postweaning decline prevented, and, if so, did lactase activity subsequently rise? Lactose was later withdrawn from the diet and then reintroduced to determine whether there were additional adaptive Received March 3, 1970. Accepted September 30, 1970. Address requests for reprints to: Dr. T. D. Bolin, The University of New South Wales School of Medicine, Prince Henry Hospital, Sydney 2036, Australia. This work was supported by grants from the National Health and Medical Research Council of Australia and the Australian Dairy Produce Board. The supplies of calcium caseinate used in the diet were provided by Mead Johnson Pty., Ltd. The statistical analysis was performed in part by Mr. A. E. Stark, M.A. (N.S.W.), Senior Lecturer, School of Human Genetics, University of New South Wales. The authors are grateful for the invaluable technical assistance of Miss Amy Ho and Mr. D. Paix, physicist, Prince Henry Hospital, Sydney, Australia. 432

responses in lactase activity. Experiments were also carried out to establish whether changes in lactase activity were associated with corresponding changes in lactose hydrolysis.

Materials and Methods Experiment 1 The effect of feeding 10% lactose on lactase activty. Wistar rats were bred and fed a completely synthetic diet containing either 10% lactose or no lactose. A synthetic diet enabled the carbohydrate content to be changed at will without affecting any of the other components. The diet consisted of 30% protein, 50% carbohydrate, and 9.6% fat with vitamin and mineral supplements. The two groups of animals were on isocaloric diets in which the total carbohydrate content was the same. Both the mother and the baby rats ate the same diet until the young rats were removed from the mother at the age of 4 weeks and were pair-fed in groups of 5. Animals were killed using ether anesthesia, in groups of 5, at intervals of 7 days until 8 weeks of age and thereafter at 2- to 3-week intervals. At 24 weeks the diets were reversed and at 32

March

1971

EFFECT OF DIET ON LACTASE ACTIVITY

weeks another group was killed. The diet was reversed once more and groups were killed at 40 and 42 weeks. Mucosal homogenates. Segments of intestine, 1 cm long, were taken from standard sites one and two-thirds of the total distance of the small intestine to obtain representative samples from jejunum and ileum. The mucosa was scraped off, immediately frozen on Dry Ice, weighed, and then homogenized in distilled water by hand in a Bellco glass homogenizer to give a final concentration of 20 mg per ml. The homogenate was immediately frozen on Dry Ice and stored at 4 C in polypropylene microcentrifuge tubes. Enzyme activity assay. Disaccharidase assays were performed on the same day under the same conditions for both test and control animals by the method of DahlqvisF at both pH 5.8 and 2.0. Protein concentration was measured by the method of Lowry et al. 8 Activity is expressed as micromoles (units) of substrate hydrolyzed per min per g of tissue protein. Alkaline phosphatase activity was estimated by the method of Bergmeyer 9 and is expressed as moles of p-nitrophenol liberated from p-nitrophenylphosphate per 30 min per mg of protein. Intestinal absorption studies. After an overnight fast, the animals were given intraperitoneal Nembutal (2 mg per 100 g of body weight) followed by ether anesthesia. The abdomen was opened and a polythene tube was inserted into the proximal end of the jejunum. At a distance of 20 cm, a second tube was inserted into the intestine, establishing an isolated loop in situ. This loop was gently washed through with about 10 ml of saline to remove residual food debris and biliary and pancreatic secretions; then a small amount of air was blown through the loop to empty it as completely as possiJ:.le. In 0.5 ml of water, 100 mg of lactose, labeled with approximately 3 f..I.C "C-l-Iactose, were injected through the proximal tube into the loop. After 20 min, the loop was washed through with 60 ml of normal saline to remove the nonabsorbed radioactivity. The loop was removed and the animal was killed. Aliquots of the standard solution and washouts were then counted in a liquid scintillation counter. The difference in activity between the two was assumed to represent hydrolysis of lactose and absorption of its constituent monosaccharides.

Experiment 2 The effect of feeding 10% liquid lactose on lactase activity. Two groups of rats were again compared under conditions similar to those in

433

experiment 1. One group was fed a diet in which the 10% lactose was given in liquid form in the drinking water and the control group had a lactose-free diet. The rats were again pair-fed in groups of 5 and killed at 2- and 3-week intervals from the age of 7 to 15 weeks.

Results Experiment 1 The rats fed the test diet containing 10% lactose grew at the same rate as those animals on the control diet. Intestinal lactase activity in animals from both groups continued to decline after weaning and reached their lowest levels at 6 weeks of age (fig. 1, table 1). Lactase activity in the control animals continued at these low levels. However, in the rats fed 10% lactose, an increase in jejunal lactase activity was first noted at the age of 8 weeks (table 1), and thereafter significantly elevated levels of intestinal lactase activity continued until the age of 24 weeks. There was also a less marked increase in ileal lactase activity occurring at a later age (table 1). The diets of two groups of animals were reversed at the age of 24 weeks. After 8 weeks it was found that there had been a decrease in lactase activity in those animals previously on a diet containing 10% lactose (fig. 1, table 1), while lactase activity rose in the animals changed to 10% lactose diet. The diets were reversed once more, and, at the end of an additional 8 weeks, jejunal lactase activity had again declined in the control animals changed back to a lactosefree diet (fig. 1, table 1). This decline was maintained at the end of 10 weeks. Although there was no increase in jejunal lactase activity in the test animals changed back to a 10% lactose diet after 8 weeks, at the end of 10 weeks lactase activity was significantly higher than in the control group. Changes in lactase activity were accompanied by changes in both sucrase and maltase activity (table 1). This change in activity was not associated with any significant alteration in alkaline phosphatase, another brush border enzyme. The increase in lactase activity in rats

434

BOLIN ET AL.

Vol. 60, No.3

JEJUNAL DISACCHARIDASES \

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4

8

12

16

20

24

28

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32

40

42

FIG. 1. Relationship of jejunal lactase activity in rats to changes in diet. The vertical arrows at 24 and 32 weeks of age indicate the changes in diet in both the test and control animals.

fed 10% lactose occurred predominantly in the enzyme assayed at pH 5.8, while there was only a small increase in that assayed at pH 2.0 (table 2) and it was not associated with an increase in mucosal protein concentration (table 3). Hydrolysis of 14C -J-lactose in the isolated loop. In vivo hydrolysis of 14C-1-lactose in 20-cm loops of intestine was compared at the age of 24 weeks in 7 control and 7 test animals. The animals fed 10% lactose hydrolyzed 44% (range, 18.5 to 70.2%) of the test dose of lactose 14C-1-lactose, while the animals fed a lactose-free diet hydrolyzed 22.4% (range, 13.0 to 30.2%) of the dose (P < 0.05). The increased hydrolysis of 14C-1lactose in animals fed 10% lactose was associated with higher levels of jejunal lactase activity (mean, 26 U per g of protein) compared with the activity in the control group (mean, 16 U per g of protein). Experiment 2 In those animals receiving lactose in liquid form, an increase in intestinal lactase activity was noted at the age of 10 weeks in both the jejunum and ileum, and these elevated levels were maintained until the age of 15 weeks when the experiment was terminated.

Discussion The postweaning decline in intestinal lactase was not prevented by feeding neonatal rats a diet containing 10% lactose. By the age of 5 weeks intestinal lactase activity was similar in both control and test animals. Continued feeding of a lactose diet produced an increase in both jejunal and ileal lactase activity. The rise was noted first in the jejunum at the age of 8 weeks, and later in the ileum, and was maintained during the period of prolonged lactose feeding. Cain et aI.,lO using rats approximately 4 weeks old with already low lactase activity, showed that lactase activity subsequently rose with continued feeding of up to 60% lactose, the rise occurring after 5 to 7 weeks on the diet. This time taken for adaptation to occur is similar to that obtained in the adult rat. l1 • 12 When there was an increase in jejunal lactase activity at 8 weeks, there was a corresponding increase in both sucrase and maltase activity. Similar alterations in sucrase and maltase accompanied the decline and subsequent rise in jejunal lactase values when lactose was withdrawn and later added to the diet. When these changes in disaccharidase activity oc-

a

(Wi)

Lac· tose

21 29

21 18

P

< 0.0025 < 0.01 < 0.4 < 0.05 < 0.01 < 0.05 < 0.015 < 0.01 < 0.01 < 0.01 < 0.01 < 0.05

P

< 0.475 < om

P

44 44

Lac-

tosefree

87

Lactose (JO",)

61 28 28 52

54

66 54 68 59 69 51 45

Lactosefree

free

Laclose-

82 83

Lactose

659 507 418 378 293 389 529 357 257 295 308 350

<0.02 NS" NS" NS" NS" <0.2 < 0.01 < 0.3 NSa <0.1 <0.01 <0.3 91 39 34 20 28 28 29 23 18 31 18 14

Lactosefree

104 49 33 19 15 21 22 19 22 37 23 26

Lac · tose

(10'";)

-

436

(10';)

Lac· tose

296

Lactosefree

Maltase

(lO'i)

Lac· tose

<0.005 24

P

16

Lactosefree

Lactase

317 262

free

Lac· tose-

418 407

Lactose

(l0',)

Maltase

free

Lac-

tose-

< 0.1 33 < 0.005 29

P

25 29

Lac· tose

P

< 0.1 < 0.49

P

< 0.025

Lactase

(lOCi )

P

< 0.4 < 0.15 < 0.45 < 0.45 < 0.0025 < 0. 15 < 0.1 < 0.1 < 0.1 < 0.25 < 0.05 < 0.01

Ie. Diets reversed between 32 and 42 weeks

< 0.1 < 0.005

P

TABLE

Sucrase

(lO',)

P

< 0.2

Jejunum

64

free

Lactose-

Sucrase

302 405 393 399 247 299 236 296 242 212 166 264

P

Lactase

lB. Diets reversed between 24 and 32 weeks

< 0.005 < 0.02 < 0.4 NS " < 0.5 <0.1 < 0.005 < 0.1 < 0.3 < 0.005 < 0.02 <0.05 TABLE

Jejunum

173 90 75 61 62 74 103 75 69 66 55 89

(10',)

tose

Lac-

P

Lactose

(l0 ',)

Maltase

Sucrase

Jejunum

fed lactose-free and 10% lactose diets

58 52

free

Lac·

tose-

71

(lO'i )

tose

Lac-

26 36 58 29 45 34 35 41 22 39 39 36

Lactosefree tose

(1O'i )

56 53

tose (lOCi)

Lac· tose-

528 413

P

NSa NSa

417 412

(lO'i)

lose

Lac-

346

Maltase

free

479

Lac-

Ileum

< 0.4

Sucrase

57

free

Lac· lose·

--

460 370 504 359 284 325 297 294 217 424 347 328

Lactose

(10';)

Lactosefree

Lactose

402 345 480 348 389 369 439 337 237 429 307 296

free

tose-

Lac·

Maltase P

< 0.1 NS" < 0.3 NS" NS" < 0.5 < 0.05 < 0.4 < 0.3 < 0.4 NSa < 0.2

P

Maltase

Sucrase

Ileum

35 36 43 27 45 39 49 36 27 34 42 52

(lO~,)

Lac-

Sucrase

Deum

lA. Intestinal disaccharidase activities: comparison of intestinal lactase, sucrase, and maltase activities in rats

< 0.025

Lactase

19

free

Lactose·

Lactase

118 37 29 15 17 26 29 24 27 26 20 30

Lac· tose (10' ;)

Lac· tosefree

29

Lactose (10' ,)

62 23 28 21 22 21 25 15 20 19 12 20

Lactosefree

Lactase

NS, not significant.

40 42

wk

Age of rat

32

wk

Age of rat

3 4 5 6 7 8 10 12 15 18 21 24

wk

Age of rat

TABLE

p

-

- --

<0.025 NSa

-

< 0.05

P

- - ---

<0.4 NS" NS" NS" < 0.1 < 0.3 < 0.01 < 0.3 NSa NS" <0.3 <0.5

P

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436

BOLIN ET AL.

2. Jejunal lactase activity at pH 5.8 and 2.0: comparison in rats fed lactose-free or 10% lactose diets

TABLE

Age of rat

Lactose-free diet

lactose diet (lOc,:)

pH 5.8

pH 2.0

pH 5.8

pH 2.0

19 23

1.6 1.5

32 33

2.0 1.7

wk

12 15

TABLE 3. Jejunal mucosal protein concentration: comparison of jejunal mucosal protein concentrations in rats fed a lactose-free or 10% lactose diet

Age of rat

Lactose-free diet

Lactose diet (JO' d

107 111 121 105 127 135 114 146 134 136 144 146 130 138 150

64 118 120 98 153 151 131 122 143 153 142 131 116 136 140

wk

3 4 5 6 7 8 10

12 15 18 21 24 32 40 42

curred, there was no alteration in another brush border enzyme, alkaline phosphatase, suggesting that the changes were specific for disaccharidases. While Broitman et al. 13 were unable to show alterations of in vivo hydrolysis of lactose accompanying a rise in lactase activity, in this study an increase in lactase activity was associated with increased in vivo hydrolysis of 14C-l-Iactose. There is evidence for the presence of two lactases in rat intestinal mucosa, one with a pH optimum of 5.5 to 6.0, and another with a pH optimum of 2 to 3.5 3, 14 The enzyme with a higher pH optimum appears to be specifically designed to hydrolyze lactose and is located in the brush border. 15 The enzyme with a more acid pH optimum seems to be less specific, probably lysosmal in origin, and is not likely to participate in intestinal digestion of lactose. The finding

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in this study of an increase in a lactase with a pH optimum of 5.8, with small alteration of the enzyme with a more acid pH optimum, is evidence for a brush border location of this enzyme, as was implied by the correlation between increased lactase activity and increased in vivo hydrolysis of lactose. It was of interest that other studies on adaptation 2, 10-12 used relatively large amounts of lactose in the diet (up to 60%), while adaptation of intestinal lactase occurred in this experiment with a level of lactose comparable with that found in the diet of normal Western children. There was also an adaptive effect of liquid lactose on lactase activity and one could speculate that milk would be just as effective. These results show that intestinal lactase in the rat is an adaptive enzyme, that adaptation is related to diet, and that changes in lactase activity are associated with corresponding changes in lactose hydrolysis. The time required for adaptation ranged between 5 and 10 weeks. However, a number of questions regarding the control of intestinal disaccharidase activity are raised. The postweaning decline in lactase activity in the rat occurs independently of lactose in the diet and presumably is dependent upon genetic control. Furthermore, lactase activity does not completely disappear in those animals who have eaten a lactose-free diet since weaning. The effect of dietary glucose on adaptation of lactase in the adult raP2 suggests that it is glucose that prevents the disappearance of intestinal lactase, while values above this are dependent upon lactose intake. The synthetic diet in this study was sucrose-free, yet normal sucrase activity was maintained. This may be due either to dietary glucose, or to the fact that control of intestinal sucrase is linked with maltase and lactase, Rosensweig and Herman 16 having previously demonstrated a link between sucrase and maltase. The interrelationship between lactase, sucrase, and maltase activities and changes in diet demonstrated in this study indicate that the dietary control of disaccharidase

March 1971

EFFECT OF DIET ON LACTASE ACTIVITY

activity is complex. Knudsen et al. 17 showed that fasting in man produced a fall in all disaccharidase activities together with protein and alkaline phosphatase. Rosensweig and Herman 18 confirmed these findings, and suggested that carbohydrate, rather than calories, was required to influence disaccharidase activity. Rosensweig and Herman 16 demonstrated that dietary sucrose, as compared with dietary glucose, significantly increased maltase and sucrase activity but not lactase activity. The concluded that fructose was the active principle in the sucrose molecule. The same workers 18 could not decrease lactase activity by feeding 4 normal subjects a 3000-cal diet for 2 months with glucose as the only carbohydrate. It would appear that the dietary control of intestinal lactase is mediated via a different mechanism than that postulated for sucrase and maltase. As the time response for adaptive changes in sucrase and maltase was coincidental with the estimated turnover time of human intestinal epithelium, it was postulated that the dietary effect on disaccharidase activity was primarily at the crypt cell level. 19 The time response for dietary adaptation of lactase in the rat consistently takes 5 to 10 weeks. 10 • 12 Moreover, relatively short term experiments at induction of lactase activity in man over periods of 10 days to 2 months have not been successful. 16. 18. 20. 21 Presumably, therefore, the effect of dietary lactose is either not primarily upon the crypt cell or there are some intermediate steps involved before the crypt cell responds. REFERENCES 1. De Groot AP, Hoogendoorn P : The detrimental

effect of lactose. II. Quantitative lactase determinations in various mammals. Netherlands Milk Dairy J 11:290- 303, 1957 2. Alvarez A, Sas J : /3-Galactosidase changes in the developing intestinal tract of the rat. Natu re (London) 190:826-827, 1961 3. Doell RG, Kretchmer N: Studies of small intestine during development. 1. Distribution and activity of /3-galactosidase. Biochim Biophys Acta 62:353-362, 1962 4. Rubino A., Zimbalatti F, Auricchio S: Intestinal

437

disaccharidase activities m adult and suckling rats. Biochim Biophys Acta 92:305-311, 1964 5. Koldovsky 0, Chytil F: Postnatal development of /3-galactosidase activity in the small intestine of the rat. Effect of adrenalectomy and diet. Biochem J 94:266-270, 1965 6. Reddy BS, Wostmann BS: Intestinal disaccharidase activities in the growing germfree and conventional rats. Arch Biochem 113:609-616, 1966 7. Dahlqvist A: Assay of intestinal disaccharidases. Anal Biochem 22:99-107, 1968 8. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurements with the Folin phenol reagent. J Bioi Chern 193:265-275, 1951 9. Bergmeyer HU: Methods of Enzymatic Analysis. Weinheim, Verlag Chemie, 1963, p 783-785 10. Cain GD, Moore P, Patterson M, et al: Stimulation of lactase by feeding lactose. Scand J Gastroent 4:545-550, 1969 11. Girardet P , Richterich R, Antener I: Adaptation de la lactase intestinale a l'administration de lactose chez Ie rat adulte. Helv Physiol Pharmacol Acta 22:7-14, 1964 12. Bolin TD, Pirola RC, Davis AE: Adaptation of intestinal lactase in the rat. Gastroenterology 57: 406-409, 1969 13. Broitman SA, Thalenfield BE, Zamcheck N: Alterations in gut lactase activity in young and adult rats fed lactose (abstr). Fed Proc 27:573, 1968 14. Dahlqvist A, Asp NG: Rat small-intestinal /3Galactosidases. Biochem J 103:86-89, 1967 15. Koldovsky 0, Heringova A, Jirsova V: /3-Galactosidase activity of the jejunum and ileum of suckling rats. Bioi Neonat 10:241-253, 1966 16. Rosensweig NS, Herman RH : Control of jejunal sucrase and maltase activity by dietary sucrose or fructose in man. A model for the study of enzyme regulation in man. J Clin Invest 47:2253- 2262, 1968 17. Knudsen KB, Bradley EM, Lecocq FR, et al: Effect of fasting and refeeding on the histology and disaccharidase activity of the human intestine. Gastroenterology 55:46-51, 1968 18. Rosensweig NS, Herman RH: Diet and disaccharidases. Amer J Clin Nutr 22:99-102, 1969 19. Rosensweig NS, Herman RH: Time response of jejunal sucrase and maltase activity to a high sucrose diet in normal man. Gastroenterology 56: 500-505, 1969 20. Newcomer AD, McGill DB: Disaccharidase activity in the small intestine: prevalence of lactase deficiency in 100 healthy subjects. Gastroenterology 53:881-889, 1967 21. Keusch GT, Troncale FJ, Thavaramara B, et al: Lactase deficiency in Thailand: effect of prolonged lactose feeding. Amer J Clin Nutr 22:638641, 1969