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Effect of chyme on mucosal enzyme levels in small intestine of the rat
Metabolism Clinical and Experimental VOL.XXI,NO.
11
NOVEMBER1972
Effect of Chyme on Mucosal Enzyme Levels in Small Intestine of the Rat By Eino Hietanen and Osmo HBnninen Partial jejunectomies, gastrojejunostomies (with closed pylorus), and jejunal Thiry-Vella loops were made in order to elucidate the role of thyme in the control of mucosal mass and the acalkaline tivities of phosphatase, ATPase, and maltase in the small intestine of the rat. After partial jejunectomy, a partially reversible mucosal hyperplasia was seen in the small intestine with the exception of distal ileum. After gastrojejunostomy a similar hyperplasia took place in the jejunum and proximal ileum. In the jejunal Thiry-Vella loops a mucosal atrophy was found in 4 wk. After partial jejunectomy the activity of alkaline phosphatase decreased slowly in 4 wk in the remaining small intestine with the duodenum as an exception. ATPase activity decreased in the duodenum. Maltase activity remained unchanged during 8 postoperative wk.
In gastrojejunostomized rats the activity of alkaline phosphatase and ATPase increased slowly during 12 wk in the jejunum aborally from the gastroenterostomy. A slight depression of maltase activity was observed in the operation area and a slight increase of enzyme activity was found in the middle of the small intestine. In jejunal Thiry-Vella loops the activity of alkaline phosphatase decreased, but no change of maltase activity could be observed during 4 wk. Perfusion of a loop with maltose solution did not cause any changes in the activity of alkaline phosphatase or maltase. The results indicate that after a change in thyme passage the adaptation takes place in the small intestine primarily by the change of mucosal mass, and at least some enzyme levels in the mucosal ceils are remarkably stable.
From the Departments of Biochemistry and Physiology, University of Turku, Turku. Finland. Received for publication February 11, 1972. Supported by USPHS Grant AM-06018-07, and a Grant from the National Research Council for Natural Sciences, Finland. Eino Hietanen, M.B.: Research Associate, Department of Physiology, University of Turku, Turku, Finland. Osmo Hiinninen, M.D., Ph.D.: Associate Professor of Biochemistry, University of Turku, Turku, Finland. Metabolism,
Vol. 21. No. 11 (November),
1972
991
992
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LTHOUGH THERE IS an almost linear decrease of the villus size,’ mucosal surface area,2 and mass3 per length unit in the small intestine from its oral to the aboral end, the mucosal enzyme activities follow in many cases a very different pattern in the rat. From the duodenum downwards there is, e.g., a rapid nonlinear decrease of the level of several enzymes related to the phosphate metabolism4j5 and glucuronide synthesiss,’ On the other hand the level of some membrane-bound digestive enzymes like maltase and other glycosidases is highest in the middle of the small intestine.“rO The experimental data which might explain these specific mucosal enzyme patterns are, however, scanty. Since the dietary modifications and certain chemical compounds cause significant changes in some mucosal enzyme 1eve1s,7,11-14 the absorption of different inducers in the different regions of the small intestine might be one explanation for the specific enzyme patterns. The normalization of the absorption of nutrients even after extensive small intestinal resections and bypasses15-1s also suggests that the intestinal mucosa has a great adaptive capacity under the influence of the natural inducers. Both the cell number and the properties of the individual cells in the mucosa may change.rg In order to elucidate the role of thyme in the control of mucosal enzyme levels in the small intestine of the rat, we have altered its normal passage by gastrointestinal surgery, and the changes in mucosal mass and some enzyme patterns have been followed. The activities of alkaline phosphatase, ATPase, and maltase have been determined, since these enzymes show different distribution patterns in the normal rats. MATERIALS
AND METHODS
Adult male Wistar rats (200-250 g at the time of operation) were fed ad lib. A total Of 200 rats were used. Before and after the operations, however, the rats were fasted overnight. Rats were anesthetized by an intraperitoneal injection of mebumal, 50 mg/kg of body weight (Sombutol, LIIke Oy, Turku, Finland), In partial jejunectomies (group A) a ZO-cm segment about 5 cm aborally from the suspensory ligament of duodenum was removed and the ends were anastomosed with a continuous silk suture.20 Gastroenterostomies (group B) were made between the antral stomach and small bowel either about 15 cm aborally from the suspensory ligament of duodenum or in the middle of the small intestine. The pylorus was closed by a silk ligature. For jejunal Thiry-Vella loops (group C) two successive lo-cm segments were cut-the first one 10 cm aborally from the suspensory ligament of duodenum-and their ends were taken through the abdominal muscles and sutured to the skin 3 cm apart. The ends of the small intestine were anastomosed as described above.20 At first a sham operation with a jejunal transection was carried out in the control rats. Since this caused no changes in the mucosal enzyme levels despite a local mucosal hyperplasia in the transection area, only a laparatomy was carried out as a routine (controls shown in the figures). No antibiotics were given to the rats. The Thiry-Vella loops were perfused daily with physiologic sodium chloride solution and in some cases with 10% maltose solution. Rats were weighed every other day during the following 3 wk. The weight gain of the partially jejunectomized and gastrojejunostomized (15 cm aborally from the suspensory ligament of duodenum) rats was as good as in the controls. The gastroenterostomy in the middle of the small intestine caused a decrease in weight with the animals dying in 1.5 mo. In order to get the tissue specimens, the rats were stunned by a blow on the head and bled by cutting renal vessels 2, 4, 8, or 12 wk after the operation. The small intestine was dissected at 4’C, and either 5- or lo-cm segments were cut out. The segments were opened
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and cleaned with a moist brush. The mucosa was scraped with an ampule file. The fresh weights (accuracy -C 5 mg) were determined immediately on a torsion balance. The samples were homogenized (10 set) in distilled water (20 times the fresh weight of the tissue sample) with the aid of an Ultra Turrax homogenizer (4°C). One milliliter of the homogenate was dried at 92°C overnight in order to determine the dry weight. Alkaline phosphatase activity was determined using p-nitrophenylphosphate as a substrate.5 ATPase activity was measured as described earlier.4,lo The maltase activity was determined as described by Dahlqvist ;21 free glucose was, however, analyzed by the o-toluidine method.22 For histologic studies about o..+cm segments from different areas of the small intestine were fixed by formaldehyde. Transverse sections were cut at 7 pm and stained with
hematoxvlin-eosin.
RESULTS Mucosal Mass The length of the small intestine of the partially jejunectomized or gastrojejunostomized rats did not increase adaptively. In the operated rats a dilation of the gut took place. The mucosal fresh weight almost doubled in 2 wk after partial jejunectomy in the small intestine both above and below the resection, with the terminal ileum as an exception (Fig. IA). This increase was reversible above the resection and also partially below it. Eight weeks after the operation the mucosal mass was significantly greater than in controls only in the jejunum aborally from the operation area (Fig. 18). In the jejunal Thiry-Vella loops a drastic decrease in mucosal fresh weight took place in 4 wk (Fig. IB). After gastrojejunostomy there was about a twofold increase in mucosal fresh weight in 2 wk immediately above and below gastroenterostomy. In the lower segments the findings were the same as 2 wk after partial
Fig. 1. The mucosal fresh weight (milligram per centimeter segment). (A) 2 wk and (B) 8 wk after a partial jejunectomy (black circles connected by solid line) in 14 and 8 rats, respectively. Segment between the vertical bars was removed. Data from 30 control rats are shown in panel (A) (black circles connected by broken line). In panel (B) the white circles indicate the fresh weights in Thiry-Vella loops 4 wk after the operation. The aboral loop was perfused daily during the last 2 wk with 1 ml of 10% maltose solution. The distance of the ileocecal valve from the pylorus has been denoted by 100. The standard errors of the means are shown. The statistical significance of the changes has been denoted by signs (-I-), p CO.05 and (+i-), p
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jejunectomy (Fig. 2A). The mucosal mass decreased gradually during the next 10 wk to the control level above but below gastroenterostomy it still remained elevated (Figs. 2B and C). The mucosal dry weight-fresh weight ratios did not change despite the marked changes in the total mucosal weights. The histologic studies revealed that the increase of mucosal mass was not due to blood congestion or leucocyte infiltration. The size of the epithelial cells, the relative number of goblet cells, and the number of mucosal villi per length unit had also remained unchanged. The mucosal thickness was, however, increased in hyperplastic areas. The decrease of the mucosal thickness and gut diameter was very marked in the jejunal Thiry-Vella loops. Enzyme Patterns After partial jejunectomy no significant changes in the activity of alkaline phosphatase were found in 2 wk after the operation (Fig. 3A). After 8 wk a slight depression had taken place with the duodenum as an exception (Fig.
(++)
:LJ+-+L +
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Fig. 2. The mucosal fresh weight (milligram per centimeter segment) (A) 2 wk, (B) 4 wk, and (C) 12 wk after a gastrojejunostomy with pyloric ligation (black circles connected by solid line). The vertical bar indicates the site of the gastroenterostomy. The number of operated rats was 5 in (A), 22 in (B), and 8 in (C). Data from 30 control rats are shown in panel (A) (black circles connected by broken
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In the jejunal Thiry-Vella loops isolated from the thyme flow, activity of alkaline phosphatase was very low 4 wk after the operation (Fig, 3B). The injection of maltose solution into the loops had no effect on enzyme activity (Fig. 3B). After gastrojejunostomy a decrease of enzyme activity in the gastroenterostomy area was observed 2 wk after the operation (Fig. 4A). Four weeks after the operation no differences between the operated and control rats could be observed (Fig. 4B), but after 12 wk a slight increase of alkaline phosphatase activity was found aborally from the gastroenterostomy (Fig. 4C). 3B).
After partial jejunectomy the activity of ATPase was significantly lowered in the duodenum 8 wk after the operation (Fig. SA). After 12 wk the enzyme activity was slightly lowered orally and increased aborally from the gastrojejunostomy (Fig. 5B). The enzyme activity was not determined in the samples from the jejunal Thiry-Vella loops. A short partial jejunectomy had no statistically significant effect on the maltase activity in the gut mucosa after 8 wk (Fig. 6A). In the jejunal ThiryVella loops the enzyme activity remained also unchanged (Fig. 6A). The injection of 1 ml of 10% maltose solution through the loop daily for 2 wk had no effect on the maltase activity in this loop (Fig. 6A). Twelve weeks after gastrojejunostomy there was a slight decrease of enzyme activity in the gastrojejunostomy area and a significant increase of activity in the middle of the gut. The maximal enzyme activity was also probably slightly shifted in the aboral direction (Fig. 6B). DISCUSSION
The thyme factors are obviously of importance in the control of the mucosal morphoIogy in the small intestine, since the mucosa1 mass almost doubles in the rat jejunum, when it is shortened or anastomosed with the stomach. Similar hyperplasia has been observed previously after small and after transposition of ileal segments to the intestinal resections23-2e was observed 2 wk after the operations, jejunum. 27 The maximal hyperplasia which confirms the previously described time course of the increase in the 25 Recently Weser and Hernandez villus height after partial jejunectomies. have shown that an extensive jejunectomy in rats causes an increase also in the number of epithehal ceIIs per unit Iength of villus.28 On the other hand, an atrophy takes place in the Thiry-Vella loops, which thyme bypasses. The mass of the duodenal and jejunal mucosa above the gastrojejunostomy does not, however, significantly reduce, which is in accordance with the hypothesis of the duodenal secretion of a villus size-increasing factor suggested by Altmann and Leblond.27 Loran and Cracker have postulated the existence of which is released at a higher rate an intestinal epithelial growth hormone,2g after intestinal resections. The mucosa contains probably also inhibitory 3s It is probable that the response of the mucosal factors of cell proliferation. mass after a change in the thyme flow is controlled in addition to the thyme factors also by these endogenous mucosal factors. An increase of small intestinal length has been shown to take place when
996
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the stimulus is strong enough (after a 75% proximal resection).2s In the present study, however, no increases of length were observed. The reason why low gastroenterostomies killed the animals after a cessation of growth remains uncertain. It is possible that the digestion of food was hampered due to a malfunction of the digestive glands or inactivation of digestive enzymes and absorption of bile acids in the long empty jejunum. One explanation could also be the poor adaptive capacity in the lower parts of the small intestine, but Nygaard 17,18 has obtained data that indicate that it is in fact greater than in the jejunum. Despite mucosal hyperplasia after partial jejunectomy and gastrojejunostomy, only small and slowly appearing changes took place in the activity of alkaline phosphatase, ATPase, and maltase. This suggests that the rapid functional adaptation previously described in connection with small intestinal resectionP’* might be due mostly to the increase of mucosal mass and the number of the mucosal cells. Thus, the amount of some enzymes at least increases primarily with the increase of cells. Tilson and Wright have reported that no changes in the total ATPase activity and a slight increase in the level of Na*, K+-activated ATPase took place in the ileum in 2 wk after an extensive small intestinal resection. a1 Weser and Hernandez have found a decrease (p < 0.05) in the specific maltase activity of the remaining small intestine28 5-6 wk after a 509'0 resection of the small intestine. After a much shorter partial jejunectomy, no statistically significant changes were observed in the maltase activity calculated on a tissue fresh weight basis in the present study. An extensive resection of the small intestine may cause changes in the water content of the mucosa and decrease the specific activity of the
1.
Fig. 3. Effect of partial jejunectomy on the activity of alkaline phosphatase in the small intestinal mucosa expressed in moles of, p-nitrophenol liberated per minute X gram of fresh tissue (A) 2 wk and (B) 8 wk after the operation [solid lines denote operated and broken line in panel (A) denotes control rats]. In panel (B) the white circles indicate the enzyme activity in Thiry-Vella loops 4 wk after the operation; the aboral loop was perfused with maltose solution. Number of operated rats was 12 in (A) and 8 in (B). Control rats numbered 14. For other explanations see Fig. 1.
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Fig. 4. The effect of gastrojejunostomy (the site is indicated by vertical bar) with pyloric ligation on the activity of alkaline phosphatase in the small intestinal mucosa expressed in moles of p-nitrophenol liberated per minute X gram of fresh tissue (A) 2 wk, (B) 4 wk, and (C) 12 wk after the operation [solid lines denote operated and broken line in panel (A) denotes control rats]. Number of operated rats was 5 in (A), 10 in (B), and 8 in (C), respectively. Control rats numbered 14. For other explanations see Fig. 1
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Fig. 5. Effect of partial jejunectomy :A) and gastrojejunostomy with pyloric igation (B) (the sites are indicated 3y vertical bars) 8 and 12 wk after the operation, respectively, on the activity ?f ATPase in the small intestinal mu:osa expressed in moles of phosphate iberated per minute X gram of fresh :issue [solid lines denote operated and )roken line in panel (A) denotes control rats]. Number of operated rats Nas eight in both cases. Control rats nlso numbered 8. For other explanations see Fig. 1.
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Fig. 6. Effect of partial jejunectomy (A) and gastrojejunostomy with pyloric ligation (B) (the sites are indicated by vertical bars) 8 and 12 wk after the operation, respectively, on the maltase activity in the small intestinal mucosa expressed in moles of glucose liberated per minute X gram of fresh tissue [solid lines denote operated and broken line in panel (A) denotes control rats]. The white circles in pane! (A) indicate the enzyme activity in Thiry-Vella loops 4 wk after the operation; the aboral loop was perfused with maltose solution daily during the last 2 wk. The number of operated rats was eight in each case. Control rats numbered 17. For other explanations see Fig. 1.
enzymes. After a 20-cm resection of the small intestine no changes in the fresh weight-dry weight ratios took place in the mucosa. The relatively high independence of the mucosal enzyme levels from the thyme flow initiates a speculation that the cells have matured before reaching the villus tips. When they meet thyme, they are specialized and have only a limited capacity to adjust their enzyme levels due to endogenous long-term enzyme repression. 32 Their genome may be masked to a great extent as is the genome in other highly specialized cells such as neurons. The short life span of the mucosal cells33 and their high level of specialization as indicated by their morphology, specific absorptive functions, and metabolic stability towards factors like thyme, apparently make them suitable models for the studies of cellular differentiation and specialization, although this had not been fully realized previously. The alterations of thyme flow caused, however, some significant changes in the enzyme levels studied. The decrease of the alkaline phosphatase activity in hyperplastic areas might be explained by a regression of cellular specialization. The activity of alkaline phosphatase is very low in the fetal gut.34 On the other hand, the slow increase of alkaline phosphatase, ATPase, and maltase activity in the jejunum after gastrojejunostomy may indicate a progress in cellular specialization to a level corresponding to the functional demands of digestion. Further evidence for the role of thyme in the control of alkaline phosphatase levels is given by Thiry-Vella loops, where alkaline phosphatase activity was considerably lowered.
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Mucosal lactase activity has been reported to increase after high lactose feeding in rats,14 which indicates that thyme factors probably control the level of this glycosidase. Data obtained in the present study indicate that even the total lack of thyme or maltose perfusions of Thiry-Vella loop have no effect on the mucosal maltase activity; the activity is apparently controlled to a great extent by endogenous factors. REFERENCES 1. Altmann, G. G., and Enesco, M.: Cell number as a measure of distribution and renewal of epithelial cells in the small intestine of growing and adult rats. Amer. J. Anat. X21:319, 1967. 2. Fisher, R. B., and Parsons, D. S.: The gradient of mucosal surface area in the small intestine of the rat. J. Anat. 84:272, 1950. 3. Hlnninen, 0.: A note on the FisherParsons surface area formula in the light of mucosal fresh and dry weights of rat small intestine. Stand. J. Gastroent. 3:110, 1968. 4. -, Hartiala, K., and Nurmikko, V.: The adenosine triphosphate contents of the mucosa and the entire wall of the alimentary tract of the rat and the activities of the enzyme system hydrolyzing ATP in the alimentary tract mucosa of the rat during the absorption of nutrients. Acta Chem. Stand. 18:937, 1964. 5. -, and Hartiala, K.: Studies on inorganic phosphate, inorganic pyrophosphatase and alkaline nonspecific phosphomonoesterase levels in the gastrointestinal tract of the rat. Acta Chem. Stand. 19:817, 1965. 6. -, Alanen, K., and Hartiala, K.: Levels of uridine diphosphate glucose dehydrogenase and UDPG in the gastrointestinal mucous membrane. Stand. J. Gastroent. 1:152, 1966. 7. -, Aitio, A., and Hartiala, K.: Gastrointestinal distribution of glucuronide synthesis and the relevant enzymes in the rat. Stand. J. Gastroent. 3:461, 1968. 8. Dahlqvist, A.: The location of carbohydratases in the digestive tract of the pig. Biochem. J. 78 :282, 1961. 9. Malhotra, 0. P., and Philip, G.: Hydrolytic enzymes of mammalian intestine. I. Distribution of hydrolytic enzymes in the goat and pig intestines. Indian J. Med. Res. 52 ~68, 1964. 10. Hietanen, E., and Hlnninen, 0.: Comparison of mucosal metabolism in the small
intestine of specific pathogen free and conventional rats. Stand. J. Gastroent. 6:127, 1971. 11. Torrontegui, G.: Hexokinase activity of intestinal mucosa after feeding different diets. Biochim. Biophys. Acta 50:164, 1961. 12. HHnninen, O., and Aitio, A.: Enhanced glucuronide formation in different tissues following drug administration. Biochem. Pharmacol. 17:2307, 1968. 13. Stifel, F. B., Rosensweig, N. S., Zakim, D., and Herman, R. H.: Dietary regulation of glycolytic enzymes. I. Adaptive changes in rat jejunum. Biochim. Biophys. Acta 170: 221, 1968. 14. Bolin, T. D., Pirola, R. C., and Davis, A. E.: Adaptation of intestinal lactase in the rat. Gastroenterology 57:406, 1969. 15. Flint, J. M.: The effect of extreme resection of the small intestine. Bull. Johns Hopkins Hosp. 22:127, 1912. 16. Althausen, T. L., Doing, R. K., Uyeyama, K., and Weiden, S.: Digestion and absorption after massive resection of the small intestine. II. Recovery of the absorptive function as shown by intestinal absorption tests in two patients and a consideration of compensatory mechanisms. Gastroenterology 16:126, 1950. 17. Nygaard, K.: Resection of the small intestine in rats. Nutritional status and adaptation of fat and protein absorption. Acta Chir. Stand. 132:731, 1966. 18. -: Resection of the small intestine in rats. II. Absorption of vitamin B12 with special regard to adaptation of absorption. Acta Chir. Stand. 1323743, 1966. 19. Weser, E.: Intestinal adaptation to small bowel resection. Amer. J. Clin. Nutr. 24:133, 1971. 20. Ethicon: Manual of Operative Procedure and Surgical Knots (ed. 12). Somerville, N. J. Ethicon Inc., 1961. 21. Dahlqvist, A.: Assay of intestinal disaccharidases. Anal. Biochem. 22~99, 1968. 22. HyvZrinen, A., and NikkilZ, A.: Spe-
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cific determination of blood glucose with toluidine. Clin. Chim. Acta 7:140, 1962.
O-
23. Booth, C. C., Evans, K. T., Menzies, T., and Street, D. F.: Intestinal hypertrophy following partial resection of the small bowel in the rat. Brit. J. Surg. 46:403, 1959. 24. Loran, M. R., and Althausen, T. L.: Cellular proliferation of intestinal epithelia in the rat two months after partial resection of the ileum. J. Biophys. Biochem. Cytol. 7~667, 1960. 25. Dowling, R. H., and Booth, C. C.: Structural and functional changes following small intestinal resection in the rat. Clin. Sci. 32:139, 1967. 26. Nygaard, K. : Morphological changes in the intestinal tract. Acta Chir. Stand. 133 :233, 1967. 27. Altmann, G. G., and Leblond, C. P.: Factors influencing villus size in the small intestine of adult rats as revealed by transposition of intestinal segments. Amer. J. Anat. 127:15, 1970. 28. Weser, E., and Hernandez, Studies of small bowel adaptation
M. H.: after in-
testinal
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in the rat. Gastroenterol-
ogy 60:69, 1971. 29. Loran, M. R., and Cracker, T. T.: Population dynamics of intestinal epithelia in the rat two months after partial resection of the ileum. J. Cell Biol. 19:285, 1963. 30. Bischoff, R.: Inhibition of mitosis by homologous tissue extracts. J. Cell Biol. 23: loA, 1964. 31. T&on, M. D., and Wright, H. K.: An adaptive change in ileal Na-K-ATPase activity after jejunectomy or jejunal transposition. Surgery 70:421, 1971. 32. HInninen, 0.: Enzyme repression. In Rechcigl, M. (Ed.): Enzyme Synthesis and Degradation in Mammalian Systems. Basel, S. Karger, 1971. 33. Bertalanffy, F. D.: Mitotic rates and renewal times of the digestive tract epithelia in the rat. Acta Anat. (Basel) 40:130, 1960. 34. Moog, F.: The functional differentiation of the small intestine. VII. Regional differences in the alkaline phosphatases of the small intestine of the mouse from birth to one year. Develop. Biol. 3:153, 1961.
11
NOVEMBER1972
Effect of Chyme on Mucosal Enzyme Levels in Small Intestine of the Rat By Eino Hietanen and Osmo HBnninen Partial jejunectomies, gastrojejunostomies (with closed pylorus), and jejunal Thiry-Vella loops were made in order to elucidate the role of thyme in the control of mucosal mass and the acalkaline tivities of phosphatase, ATPase, and maltase in the small intestine of the rat. After partial jejunectomy, a partially reversible mucosal hyperplasia was seen in the small intestine with the exception of distal ileum. After gastrojejunostomy a similar hyperplasia took place in the jejunum and proximal ileum. In the jejunal Thiry-Vella loops a mucosal atrophy was found in 4 wk. After partial jejunectomy the activity of alkaline phosphatase decreased slowly in 4 wk in the remaining small intestine with the duodenum as an exception. ATPase activity decreased in the duodenum. Maltase activity remained unchanged during 8 postoperative wk.
In gastrojejunostomized rats the activity of alkaline phosphatase and ATPase increased slowly during 12 wk in the jejunum aborally from the gastroenterostomy. A slight depression of maltase activity was observed in the operation area and a slight increase of enzyme activity was found in the middle of the small intestine. In jejunal Thiry-Vella loops the activity of alkaline phosphatase decreased, but no change of maltase activity could be observed during 4 wk. Perfusion of a loop with maltose solution did not cause any changes in the activity of alkaline phosphatase or maltase. The results indicate that after a change in thyme passage the adaptation takes place in the small intestine primarily by the change of mucosal mass, and at least some enzyme levels in the mucosal ceils are remarkably stable.
From the Departments of Biochemistry and Physiology, University of Turku, Turku. Finland. Received for publication February 11, 1972. Supported by USPHS Grant AM-06018-07, and a Grant from the National Research Council for Natural Sciences, Finland. Eino Hietanen, M.B.: Research Associate, Department of Physiology, University of Turku, Turku, Finland. Osmo Hiinninen, M.D., Ph.D.: Associate Professor of Biochemistry, University of Turku, Turku, Finland. Metabolism,
Vol. 21. No. 11 (November),
1972
991
992
HIETANEN
AND HANNINEN
A
LTHOUGH THERE IS an almost linear decrease of the villus size,’ mucosal surface area,2 and mass3 per length unit in the small intestine from its oral to the aboral end, the mucosal enzyme activities follow in many cases a very different pattern in the rat. From the duodenum downwards there is, e.g., a rapid nonlinear decrease of the level of several enzymes related to the phosphate metabolism4j5 and glucuronide synthesiss,’ On the other hand the level of some membrane-bound digestive enzymes like maltase and other glycosidases is highest in the middle of the small intestine.“rO The experimental data which might explain these specific mucosal enzyme patterns are, however, scanty. Since the dietary modifications and certain chemical compounds cause significant changes in some mucosal enzyme 1eve1s,7,11-14 the absorption of different inducers in the different regions of the small intestine might be one explanation for the specific enzyme patterns. The normalization of the absorption of nutrients even after extensive small intestinal resections and bypasses15-1s also suggests that the intestinal mucosa has a great adaptive capacity under the influence of the natural inducers. Both the cell number and the properties of the individual cells in the mucosa may change.rg In order to elucidate the role of thyme in the control of mucosal enzyme levels in the small intestine of the rat, we have altered its normal passage by gastrointestinal surgery, and the changes in mucosal mass and some enzyme patterns have been followed. The activities of alkaline phosphatase, ATPase, and maltase have been determined, since these enzymes show different distribution patterns in the normal rats. MATERIALS
AND METHODS
Adult male Wistar rats (200-250 g at the time of operation) were fed ad lib. A total Of 200 rats were used. Before and after the operations, however, the rats were fasted overnight. Rats were anesthetized by an intraperitoneal injection of mebumal, 50 mg/kg of body weight (Sombutol, LIIke Oy, Turku, Finland), In partial jejunectomies (group A) a ZO-cm segment about 5 cm aborally from the suspensory ligament of duodenum was removed and the ends were anastomosed with a continuous silk suture.20 Gastroenterostomies (group B) were made between the antral stomach and small bowel either about 15 cm aborally from the suspensory ligament of duodenum or in the middle of the small intestine. The pylorus was closed by a silk ligature. For jejunal Thiry-Vella loops (group C) two successive lo-cm segments were cut-the first one 10 cm aborally from the suspensory ligament of duodenum-and their ends were taken through the abdominal muscles and sutured to the skin 3 cm apart. The ends of the small intestine were anastomosed as described above.20 At first a sham operation with a jejunal transection was carried out in the control rats. Since this caused no changes in the mucosal enzyme levels despite a local mucosal hyperplasia in the transection area, only a laparatomy was carried out as a routine (controls shown in the figures). No antibiotics were given to the rats. The Thiry-Vella loops were perfused daily with physiologic sodium chloride solution and in some cases with 10% maltose solution. Rats were weighed every other day during the following 3 wk. The weight gain of the partially jejunectomized and gastrojejunostomized (15 cm aborally from the suspensory ligament of duodenum) rats was as good as in the controls. The gastroenterostomy in the middle of the small intestine caused a decrease in weight with the animals dying in 1.5 mo. In order to get the tissue specimens, the rats were stunned by a blow on the head and bled by cutting renal vessels 2, 4, 8, or 12 wk after the operation. The small intestine was dissected at 4’C, and either 5- or lo-cm segments were cut out. The segments were opened
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and cleaned with a moist brush. The mucosa was scraped with an ampule file. The fresh weights (accuracy -C 5 mg) were determined immediately on a torsion balance. The samples were homogenized (10 set) in distilled water (20 times the fresh weight of the tissue sample) with the aid of an Ultra Turrax homogenizer (4°C). One milliliter of the homogenate was dried at 92°C overnight in order to determine the dry weight. Alkaline phosphatase activity was determined using p-nitrophenylphosphate as a substrate.5 ATPase activity was measured as described earlier.4,lo The maltase activity was determined as described by Dahlqvist ;21 free glucose was, however, analyzed by the o-toluidine method.22 For histologic studies about o..+cm segments from different areas of the small intestine were fixed by formaldehyde. Transverse sections were cut at 7 pm and stained with
hematoxvlin-eosin.
RESULTS Mucosal Mass The length of the small intestine of the partially jejunectomized or gastrojejunostomized rats did not increase adaptively. In the operated rats a dilation of the gut took place. The mucosal fresh weight almost doubled in 2 wk after partial jejunectomy in the small intestine both above and below the resection, with the terminal ileum as an exception (Fig. IA). This increase was reversible above the resection and also partially below it. Eight weeks after the operation the mucosal mass was significantly greater than in controls only in the jejunum aborally from the operation area (Fig. 18). In the jejunal Thiry-Vella loops a drastic decrease in mucosal fresh weight took place in 4 wk (Fig. IB). After gastrojejunostomy there was about a twofold increase in mucosal fresh weight in 2 wk immediately above and below gastroenterostomy. In the lower segments the findings were the same as 2 wk after partial
Fig. 1. The mucosal fresh weight (milligram per centimeter segment). (A) 2 wk and (B) 8 wk after a partial jejunectomy (black circles connected by solid line) in 14 and 8 rats, respectively. Segment between the vertical bars was removed. Data from 30 control rats are shown in panel (A) (black circles connected by broken line). In panel (B) the white circles indicate the fresh weights in Thiry-Vella loops 4 wk after the operation. The aboral loop was perfused daily during the last 2 wk with 1 ml of 10% maltose solution. The distance of the ileocecal valve from the pylorus has been denoted by 100. The standard errors of the means are shown. The statistical significance of the changes has been denoted by signs (-I-), p CO.05 and (+i-), p
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jejunectomy (Fig. 2A). The mucosal mass decreased gradually during the next 10 wk to the control level above but below gastroenterostomy it still remained elevated (Figs. 2B and C). The mucosal dry weight-fresh weight ratios did not change despite the marked changes in the total mucosal weights. The histologic studies revealed that the increase of mucosal mass was not due to blood congestion or leucocyte infiltration. The size of the epithelial cells, the relative number of goblet cells, and the number of mucosal villi per length unit had also remained unchanged. The mucosal thickness was, however, increased in hyperplastic areas. The decrease of the mucosal thickness and gut diameter was very marked in the jejunal Thiry-Vella loops. Enzyme Patterns After partial jejunectomy no significant changes in the activity of alkaline phosphatase were found in 2 wk after the operation (Fig. 3A). After 8 wk a slight depression had taken place with the duodenum as an exception (Fig.
(++)
:LJ+-+L +
0
50
Fig. 2. The mucosal fresh weight (milligram per centimeter segment) (A) 2 wk, (B) 4 wk, and (C) 12 wk after a gastrojejunostomy with pyloric ligation (black circles connected by solid line). The vertical bar indicates the site of the gastroenterostomy. The number of operated rats was 5 in (A), 22 in (B), and 8 in (C). Data from 30 control rats are shown in panel (A) (black circles connected by broken
MUCOSAL
ENZYME
LEVELS
995
In the jejunal Thiry-Vella loops isolated from the thyme flow, activity of alkaline phosphatase was very low 4 wk after the operation (Fig, 3B). The injection of maltose solution into the loops had no effect on enzyme activity (Fig. 3B). After gastrojejunostomy a decrease of enzyme activity in the gastroenterostomy area was observed 2 wk after the operation (Fig. 4A). Four weeks after the operation no differences between the operated and control rats could be observed (Fig. 4B), but after 12 wk a slight increase of alkaline phosphatase activity was found aborally from the gastroenterostomy (Fig. 4C). 3B).
After partial jejunectomy the activity of ATPase was significantly lowered in the duodenum 8 wk after the operation (Fig. SA). After 12 wk the enzyme activity was slightly lowered orally and increased aborally from the gastrojejunostomy (Fig. 5B). The enzyme activity was not determined in the samples from the jejunal Thiry-Vella loops. A short partial jejunectomy had no statistically significant effect on the maltase activity in the gut mucosa after 8 wk (Fig. 6A). In the jejunal ThiryVella loops the enzyme activity remained also unchanged (Fig. 6A). The injection of 1 ml of 10% maltose solution through the loop daily for 2 wk had no effect on the maltase activity in this loop (Fig. 6A). Twelve weeks after gastrojejunostomy there was a slight decrease of enzyme activity in the gastrojejunostomy area and a significant increase of activity in the middle of the gut. The maximal enzyme activity was also probably slightly shifted in the aboral direction (Fig. 6B). DISCUSSION
The thyme factors are obviously of importance in the control of the mucosal morphoIogy in the small intestine, since the mucosa1 mass almost doubles in the rat jejunum, when it is shortened or anastomosed with the stomach. Similar hyperplasia has been observed previously after small and after transposition of ileal segments to the intestinal resections23-2e was observed 2 wk after the operations, jejunum. 27 The maximal hyperplasia which confirms the previously described time course of the increase in the 25 Recently Weser and Hernandez villus height after partial jejunectomies. have shown that an extensive jejunectomy in rats causes an increase also in the number of epithehal ceIIs per unit Iength of villus.28 On the other hand, an atrophy takes place in the Thiry-Vella loops, which thyme bypasses. The mass of the duodenal and jejunal mucosa above the gastrojejunostomy does not, however, significantly reduce, which is in accordance with the hypothesis of the duodenal secretion of a villus size-increasing factor suggested by Altmann and Leblond.27 Loran and Cracker have postulated the existence of which is released at a higher rate an intestinal epithelial growth hormone,2g after intestinal resections. The mucosa contains probably also inhibitory 3s It is probable that the response of the mucosal factors of cell proliferation. mass after a change in the thyme flow is controlled in addition to the thyme factors also by these endogenous mucosal factors. An increase of small intestinal length has been shown to take place when
996
HIETANEN
AND HANNINEN
the stimulus is strong enough (after a 75% proximal resection).2s In the present study, however, no increases of length were observed. The reason why low gastroenterostomies killed the animals after a cessation of growth remains uncertain. It is possible that the digestion of food was hampered due to a malfunction of the digestive glands or inactivation of digestive enzymes and absorption of bile acids in the long empty jejunum. One explanation could also be the poor adaptive capacity in the lower parts of the small intestine, but Nygaard 17,18 has obtained data that indicate that it is in fact greater than in the jejunum. Despite mucosal hyperplasia after partial jejunectomy and gastrojejunostomy, only small and slowly appearing changes took place in the activity of alkaline phosphatase, ATPase, and maltase. This suggests that the rapid functional adaptation previously described in connection with small intestinal resectionP’* might be due mostly to the increase of mucosal mass and the number of the mucosal cells. Thus, the amount of some enzymes at least increases primarily with the increase of cells. Tilson and Wright have reported that no changes in the total ATPase activity and a slight increase in the level of Na*, K+-activated ATPase took place in the ileum in 2 wk after an extensive small intestinal resection. a1 Weser and Hernandez have found a decrease (p < 0.05) in the specific maltase activity of the remaining small intestine28 5-6 wk after a 509'0 resection of the small intestine. After a much shorter partial jejunectomy, no statistically significant changes were observed in the maltase activity calculated on a tissue fresh weight basis in the present study. An extensive resection of the small intestine may cause changes in the water content of the mucosa and decrease the specific activity of the
1.
Fig. 3. Effect of partial jejunectomy on the activity of alkaline phosphatase in the small intestinal mucosa expressed in moles of, p-nitrophenol liberated per minute X gram of fresh tissue (A) 2 wk and (B) 8 wk after the operation [solid lines denote operated and broken line in panel (A) denotes control rats]. In panel (B) the white circles indicate the enzyme activity in Thiry-Vella loops 4 wk after the operation; the aboral loop was perfused with maltose solution. Number of operated rats was 12 in (A) and 8 in (B). Control rats numbered 14. For other explanations see Fig. 1.
MUCOSAL ENZYME LEVELS
997
Fig. 4. The effect of gastrojejunostomy (the site is indicated by vertical bar) with pyloric ligation on the activity of alkaline phosphatase in the small intestinal mucosa expressed in moles of p-nitrophenol liberated per minute X gram of fresh tissue (A) 2 wk, (B) 4 wk, and (C) 12 wk after the operation [solid lines denote operated and broken line in panel (A) denotes control rats]. Number of operated rats was 5 in (A), 10 in (B), and 8 in (C), respectively. Control rats numbered 14. For other explanations see Fig. 1
I
0 4
0
50
.E
A
,
lo(
Fig. 5. Effect of partial jejunectomy :A) and gastrojejunostomy with pyloric igation (B) (the sites are indicated 3y vertical bars) 8 and 12 wk after the operation, respectively, on the activity ?f ATPase in the small intestinal mu:osa expressed in moles of phosphate iberated per minute X gram of fresh :issue [solid lines denote operated and )roken line in panel (A) denotes control rats]. Number of operated rats Nas eight in both cases. Control rats nlso numbered 8. For other explanations see Fig. 1.
HIETANEN
A
0
cn4 x .;
2 z2 v\ b
6
0
AND HANNINEN
Fig. 6. Effect of partial jejunectomy (A) and gastrojejunostomy with pyloric ligation (B) (the sites are indicated by vertical bars) 8 and 12 wk after the operation, respectively, on the maltase activity in the small intestinal mucosa expressed in moles of glucose liberated per minute X gram of fresh tissue [solid lines denote operated and broken line in panel (A) denotes control rats]. The white circles in pane! (A) indicate the enzyme activity in Thiry-Vella loops 4 wk after the operation; the aboral loop was perfused with maltose solution daily during the last 2 wk. The number of operated rats was eight in each case. Control rats numbered 17. For other explanations see Fig. 1.
enzymes. After a 20-cm resection of the small intestine no changes in the fresh weight-dry weight ratios took place in the mucosa. The relatively high independence of the mucosal enzyme levels from the thyme flow initiates a speculation that the cells have matured before reaching the villus tips. When they meet thyme, they are specialized and have only a limited capacity to adjust their enzyme levels due to endogenous long-term enzyme repression. 32 Their genome may be masked to a great extent as is the genome in other highly specialized cells such as neurons. The short life span of the mucosal cells33 and their high level of specialization as indicated by their morphology, specific absorptive functions, and metabolic stability towards factors like thyme, apparently make them suitable models for the studies of cellular differentiation and specialization, although this had not been fully realized previously. The alterations of thyme flow caused, however, some significant changes in the enzyme levels studied. The decrease of the alkaline phosphatase activity in hyperplastic areas might be explained by a regression of cellular specialization. The activity of alkaline phosphatase is very low in the fetal gut.34 On the other hand, the slow increase of alkaline phosphatase, ATPase, and maltase activity in the jejunum after gastrojejunostomy may indicate a progress in cellular specialization to a level corresponding to the functional demands of digestion. Further evidence for the role of thyme in the control of alkaline phosphatase levels is given by Thiry-Vella loops, where alkaline phosphatase activity was considerably lowered.
MUCOSAL
ENZYME
LEVELS
999
Mucosal lactase activity has been reported to increase after high lactose feeding in rats,14 which indicates that thyme factors probably control the level of this glycosidase. Data obtained in the present study indicate that even the total lack of thyme or maltose perfusions of Thiry-Vella loop have no effect on the mucosal maltase activity; the activity is apparently controlled to a great extent by endogenous factors. REFERENCES 1. Altmann, G. G., and Enesco, M.: Cell number as a measure of distribution and renewal of epithelial cells in the small intestine of growing and adult rats. Amer. J. Anat. X21:319, 1967. 2. Fisher, R. B., and Parsons, D. S.: The gradient of mucosal surface area in the small intestine of the rat. J. Anat. 84:272, 1950. 3. Hlnninen, 0.: A note on the FisherParsons surface area formula in the light of mucosal fresh and dry weights of rat small intestine. Stand. J. Gastroent. 3:110, 1968. 4. -, Hartiala, K., and Nurmikko, V.: The adenosine triphosphate contents of the mucosa and the entire wall of the alimentary tract of the rat and the activities of the enzyme system hydrolyzing ATP in the alimentary tract mucosa of the rat during the absorption of nutrients. Acta Chem. Stand. 18:937, 1964. 5. -, and Hartiala, K.: Studies on inorganic phosphate, inorganic pyrophosphatase and alkaline nonspecific phosphomonoesterase levels in the gastrointestinal tract of the rat. Acta Chem. Stand. 19:817, 1965. 6. -, Alanen, K., and Hartiala, K.: Levels of uridine diphosphate glucose dehydrogenase and UDPG in the gastrointestinal mucous membrane. Stand. J. Gastroent. 1:152, 1966. 7. -, Aitio, A., and Hartiala, K.: Gastrointestinal distribution of glucuronide synthesis and the relevant enzymes in the rat. Stand. J. Gastroent. 3:461, 1968. 8. Dahlqvist, A.: The location of carbohydratases in the digestive tract of the pig. Biochem. J. 78 :282, 1961. 9. Malhotra, 0. P., and Philip, G.: Hydrolytic enzymes of mammalian intestine. I. Distribution of hydrolytic enzymes in the goat and pig intestines. Indian J. Med. Res. 52 ~68, 1964. 10. Hietanen, E., and Hlnninen, 0.: Comparison of mucosal metabolism in the small
intestine of specific pathogen free and conventional rats. Stand. J. Gastroent. 6:127, 1971. 11. Torrontegui, G.: Hexokinase activity of intestinal mucosa after feeding different diets. Biochim. Biophys. Acta 50:164, 1961. 12. HHnninen, O., and Aitio, A.: Enhanced glucuronide formation in different tissues following drug administration. Biochem. Pharmacol. 17:2307, 1968. 13. Stifel, F. B., Rosensweig, N. S., Zakim, D., and Herman, R. H.: Dietary regulation of glycolytic enzymes. I. Adaptive changes in rat jejunum. Biochim. Biophys. Acta 170: 221, 1968. 14. Bolin, T. D., Pirola, R. C., and Davis, A. E.: Adaptation of intestinal lactase in the rat. Gastroenterology 57:406, 1969. 15. Flint, J. M.: The effect of extreme resection of the small intestine. Bull. Johns Hopkins Hosp. 22:127, 1912. 16. Althausen, T. L., Doing, R. K., Uyeyama, K., and Weiden, S.: Digestion and absorption after massive resection of the small intestine. II. Recovery of the absorptive function as shown by intestinal absorption tests in two patients and a consideration of compensatory mechanisms. Gastroenterology 16:126, 1950. 17. Nygaard, K.: Resection of the small intestine in rats. Nutritional status and adaptation of fat and protein absorption. Acta Chir. Stand. 132:731, 1966. 18. -: Resection of the small intestine in rats. II. Absorption of vitamin B12 with special regard to adaptation of absorption. Acta Chir. Stand. 1323743, 1966. 19. Weser, E.: Intestinal adaptation to small bowel resection. Amer. J. Clin. Nutr. 24:133, 1971. 20. Ethicon: Manual of Operative Procedure and Surgical Knots (ed. 12). Somerville, N. J. Ethicon Inc., 1961. 21. Dahlqvist, A.: Assay of intestinal disaccharidases. Anal. Biochem. 22~99, 1968. 22. HyvZrinen, A., and NikkilZ, A.: Spe-
1000
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cific determination of blood glucose with toluidine. Clin. Chim. Acta 7:140, 1962.
O-
23. Booth, C. C., Evans, K. T., Menzies, T., and Street, D. F.: Intestinal hypertrophy following partial resection of the small bowel in the rat. Brit. J. Surg. 46:403, 1959. 24. Loran, M. R., and Althausen, T. L.: Cellular proliferation of intestinal epithelia in the rat two months after partial resection of the ileum. J. Biophys. Biochem. Cytol. 7~667, 1960. 25. Dowling, R. H., and Booth, C. C.: Structural and functional changes following small intestinal resection in the rat. Clin. Sci. 32:139, 1967. 26. Nygaard, K. : Morphological changes in the intestinal tract. Acta Chir. Stand. 133 :233, 1967. 27. Altmann, G. G., and Leblond, C. P.: Factors influencing villus size in the small intestine of adult rats as revealed by transposition of intestinal segments. Amer. J. Anat. 127:15, 1970. 28. Weser, E., and Hernandez, Studies of small bowel adaptation
M. H.: after in-
testinal
resection
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in the rat. Gastroenterol-
ogy 60:69, 1971. 29. Loran, M. R., and Cracker, T. T.: Population dynamics of intestinal epithelia in the rat two months after partial resection of the ileum. J. Cell Biol. 19:285, 1963. 30. Bischoff, R.: Inhibition of mitosis by homologous tissue extracts. J. Cell Biol. 23: loA, 1964. 31. T&on, M. D., and Wright, H. K.: An adaptive change in ileal Na-K-ATPase activity after jejunectomy or jejunal transposition. Surgery 70:421, 1971. 32. HInninen, 0.: Enzyme repression. In Rechcigl, M. (Ed.): Enzyme Synthesis and Degradation in Mammalian Systems. Basel, S. Karger, 1971. 33. Bertalanffy, F. D.: Mitotic rates and renewal times of the digestive tract epithelia in the rat. Acta Anat. (Basel) 40:130, 1960. 34. Moog, F.: The functional differentiation of the small intestine. VII. Regional differences in the alkaline phosphatases of the small intestine of the mouse from birth to one year. Develop. Biol. 3:153, 1961.