/ . Biochem., 78, 475-480 (1975)
Circadian Rhythms in Digestive Enzymes in the Small Intestine of Rats I. Patterns of the Rhythms in Various Regions of the Small Intestine1 Masayuki SAITO, Eiko MURAKAMI, Teruo NISHIDA, Yoshiki FUJISAWA, and Masami SUDA Department of Medical Chemistry, School of Medicine, Ehime University, Shigenobu, Ehime 791-02 Received for publication, April 14, 1975
The activities of the digestive enzymes, maltase [EC 3.2.1.20], sucrase [EC 3.2.1.26], trehalase [EC 3.2.1.28], leucine aminopeptidase [EC 3.4.11.1], and alkaline phosphatase [EC 3.1.3.1] were measured in various regions of the small intestine of rats. The activities of all these enzymes were much higher in the jejunum than in the ileum, and in the distal regions of the ileum no sucrase, trehalase or alkaline phosphatase activity was detected. In the jejunum, the activities of all the enzymes tested exhibited clear circadian variations with the highest activity at 0000—0400 h and the lowest at 1200 h when the rats were fed ad libitum. In the ileum, maltase and sucrase also exhibited circadian variations, but the amplitude of the rhythm was smaller than that in the jejunum. Trehalase and alkaline phosphatase did not show any circadian variation in the ileum. Leucine aminopeptidase showed a circadian variation in the ileum with the same amplitude as in the jejunum. The phase of the circadian variations shifted about half a day when the rats were fed in the daytime, but the amplitude of the rhythm did not change.
Biochemical and histochemical studies have indicated that the epithelial cells of the small intestine bind many hydrolytic enzymes, such as maltase [EC £ 2.1.20], sucrase [EC 3.2.1.26], trehalase [EC 3.2.1.28], lactase [EC 3.2.1.23], dipeptidases, leucine aminopeptidase [EC 3.4.11.1], and alkaline phosphatase [EC 3.1.3.1]. Some of these enyzmes have been suggested to be important in the final stage of digestion or absorption of nutrients (/, 2). We reported previously that the activities 1
This study was partly supported by grants from the Yamanouchi Foundation of Metabolic Diseases. Vol. 78, No. 3, 1975
of maltase and leucine aminopeptidase in the entire small intestine of rats exhibited circadian fluctuations, and that the phase of these circadian rhythms was shifted by changing the feeding time (3). The circadian fluctuations in the activities of some hydrolases in rat small intestine were also demonstrated histochemically by Schafer et al. (4). Other investigations have shown circadian rhythmic changes in the movement of isolated intestine (5), active trransport of L-histidine (6") and the mitotic activity of the epithelium (7). These rhythms in the small intestine seem to have a close relationship to the cycle of food
475
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M. SAITO, E. MURAKAMI, T. NISHIDA, Y. FUJISAWA, and M. SUDA
intake, but few data are available on their mechanisms or physiological significance. It is well-known that digestive and absorptive activities vary in different regions of the small intestine (8). For example, disaccharidase activities are highest in the jejunum and lower in the ileum in rats, and so the jejunum may be the site of the most active hydrolysis of disaccharides. This region is also likely to be the site of most active absorption of disaccharides or the monosaccharides liberated from them. Thus, it seems likely that the circadian variation in disaccharidase should be clearer in the jejunum than in the ileum. In this work, we examined the rhythmic changes in the activities of maltase, sucrase, trehalase, leucine aminopeptidase, and alkaline phosphatase in different regions of the small intestine of rats. The effect on the rhythm of altering the feeding time was also studied. EXPERIMENTAL PROCEDURE Male Wistar strain rats (150-250 g) were kept in wire-bottomed cages at 22±2° under a conventional lighting regime with a dark night. The rats were fed on laboratory chow (Oriental Yeast Co.) and allowed free access to water. In one experiment, rats were fed ad libitum, and in the other they were fed from 0900 to 1500 h. Rats were decapitated at 4 hr intervals, and their entire small intestine was rapidly removed, and divided into six segments of equal length, which were numbered serially from the duodenal region. Each segment was washed out with cold 0.9% NaCl to remove the contents. Then the mucosa was scraped off with a glass slide and homogenized with about 10 volumes of 10 mM sodium phosphate buffer (pH 7.0) using a Teflon pestled homogenizer. Disaccharidase activities were determined by a slight modification of Dahlqvist's method (9). The assay was performed in a reaction mixture containing 40 mM maltose, sucrose or trehalose, 80 mM sodium phosphate buffer (pH 7.0) and homogenates (10-50 fig protein) in a total volume of 0.5 ml at 37° for 10—30 min. The glucose liberated was estimated using specific glucose oxidase [EC
1.1.3.4] reagents (Worthington Biochemical Co.). Leucine aminopeptidase activity was measured in a reaction mixture containing 1 mM L-leucyl-j3-naphthylamide, 1 mM CoCl2, 80 mM sodium phosphate buffer (pH 7.0), and homogenates (10—50 fig protein) in a total volume of 0.5 ml. After incubation at 37° for 10 min, the reaction was terminated by adding 0.5 ml of 10% trichloroacetic acid, and the (3naphthylamine formed was measured by the method of Goldberg and Rutenberg {10). Alkaline phosphatase activity was determined under the conditions described by Forstner et al. (11). The assay was performed in a reaction mixture containing 4 mM />-nitrophenyl phosphate, 4 mM MgCl2, 80 mM sodium glycine buffer (pH 9.5), and homogenates (10—50 fig protein) in a total volume of 0.5 ml at 37° for 2-10 min. After adding 2 ml of 0.1 N NaOH, free ^-nitrophenol was determined spectrophotometrically at 400 nm. Protein was estimated by the method of Lowry et al. with bovine serum albumin as a standard (12). RESULTS The distributions of the activities of maltase, sucrase, trehalase, leucine aminopeptidase, and alkaline phosphatase were examined in the small intestines of five rats. In this experiment, the rats were fed ad libitum and killed at 2000 h. As described in the "EXPERIMENTAL PROCEDURE," the entire small intestine was divided into six equal segments, which were numbered serially from the duodenal region. Thus, segments 2 and 6 roughly correspond to the proximal part of the jejunum and the distal part of the ileum, respectively. Sucrase activity was highest in segment 3, being significantly lower in segment 1, and also in segments 4 through 6, which showed a progressive decrease in activity, as indicated in Table I. The distribution of maltase activity was essentially the same as that of sucrase activity, although it seems that there was a significant difference between the activities in segments 3 and 4. Leucine aminopeptidase activity was found to be lower in segments 1 and 6 than in segments 2, 3, 4, and 5, the latter having similar activities. The / . Biochem.
CIRCADIAN RHYTHMS IN RAT INTESTINAL DIGESTIVE ENZYMES
477
TABLE I. Distributions of enzyme activities in the small intestine. Rats were fed ad libium, and decapitated at 2000 h. The entire small intestine was divided into six equal segments, which were numbered serially from the duodenal region. Values are the means of those in five rats, with the standard errors. Enzyme activity (/umoles of product formed per min per mg protein) Segment No. 1 2 3 4 5 6 a
Maltase
Sucrase
Trehalase
Alkaline phosphatase
Leucine aminopeptidase
0.804+0.047 0.911 ±0.035 1.10 ±0.07 1.08 ±0.03 0.698 ±0.017 0.224+0.013
0.078 ±0.002 0.130 ±0. 007 0.142 ±0.013 0.098+0.004 0.031 ±0.003 N.D."
0.114+0.001 0.093+0.003 0.059+0. 006 0.029 ±0.004 0.006 ±0.001 N.D.
1.05 +0.09 0.471+0.027 0.197+0.028 0.071+0.028 0.023+0.015 N.D.
0.053 ±0.003 0.086 ±0.003 0.089 ±0.006 0.097 ±0.006 0. 087 ±0.004 0.050 ±0.003
Not Detectable.
distribution of trehalase activity was essentially the same as that of alkaline phosphatase activity, being different from those of sucrase, maltase, and leucine aminopeptidase. The activities of trehalase and alkaline phosphatase were both highest in segment 1, and decreased progressively in segments 2 through 5, no activity being detectable in segment 6. In Table I, the enzyme activities are expressed as specific activities, i.e., ^moles of product formed per min per mg protein, and the total activity per segment is roughly proportional to the specific activity, since the protein content per segment was found to be nearly the same among segments. To examine the circadian variations of the enzyme activities in various regions of the small intestine, rats were fed ad libitum and killed at 4 hr intervals, and their enzyme activities were measured in segments 2, 4, and 6. As shown inFig. 1, maltase_ activity exhibited a clear circadian variation in all three segments. Activity was highest at 0000 h and lowest at 1200 h in all segments. The ratio of the highest to the lowest activity, i.e. the amplitude of the rhythm, was about 2.6 in segment 2, which was significantly higher than those in segments 4 and 6 (1.8 and 1.7, respectively). Sucrase activity also showed a circadian fluctuation in segments 2 and 4, with the highest activity at 0000 h and the lowest at 1200 h, and the ratio of the former to the Vol. 78, No. 3, 1975
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Fig. 1. Circadian variations of mallase activity in various regions of the small intestine. Rats were fed ad libitum, and decapitated at 4 hr intervals. Segments of the small intestine were obtained as for Table I, and maltase activity was measured in homogenates of the mucosa of each segment. Values are the means of those in five rats, with the standard errors. O, segment 2; 0 , segment 4; x , segment 6.
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M. SAITO, E. MURAKAMI, T. NISHIDA, Y. FUJISAWA, and M. SUDA
latter was 2.0 in segment 2, which was definitely higher than that of 1.5 in segment 4 (data not shown). In segment 6, no activity was detected at any time of the day. Figure 2 shows the circadian fluctuations of alkaline phosphatase in segments 2 and 4. In segment 2, the activity exhibited a clear circadian variation with the same pattern as those of maltase and sucrase, and the amplitude of the rhythm was 2.1. However, in segment 4, the activity showed no circadian variation. Segment 4 also showed no circadian variation in trehalase activity, which exhibited a clear circadian variation in segment 2 with the same pattern as those of alkaline phosphatase, maltase, and sucrase. In segment 6, no activity of alkaline phosphatase or trehalase was detected at any time of the day. Leucine aminopeptidase activity also showed a clear circadian variation with the highest activity at 0400 h in all segments examined, as shown in Fig. 3. However, there was no significant difference in the amplitudes of the rhythms in segments 2, 4, and 6, unlike in
the cases of maltase, sucrase, alkaline phosphatase, and trehalase. In summary, all the enzyme activities tested exhibited clear circadian fluctuations in the small intestine with the highest activities 0000-0400 h and the lowest at 1200 h. The amplitudes of the rhythms of disaccharidases and alkaline phosphatase were highest in segment 2, corresponding to the proximal region of the jejunum, and the rhythmic variation was less marked or not detected in segments 4 and 6, corresponding to the middle region of the small intestine and the distal region of the ileum, respectively. Rats are nocturnal, so they eat during the night when they are fed ad libitum. As described above, the enzyme activities in the intestine were found to be higher at night. Next, the relationship between the rhythms of the enzyme activities and food intake was examined. Rats were fed during the daytime from 0900 h to 1500 h every day for 2 weeks, and kept under a conventional lighting schedule
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Fig. 2. Circadian variations of alkaline phosphatase activity in various regions of the small intestine. Alkaline phosphatase activity was measured in the same homogenate used for Fig. 1. Values are the means of those in five rats, with the standard errors. O, segment 2; • , segment 4.
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Fig. 3. Circadian variations of leucine aminopeptidase activity in various regions of the small intestine. Leucine aminopeptidase activity was measured in the same homogenate used for Fig. 1. Values are the means of those in five rats, with the standard errors. O, segment 2; • , segment 4; x, segment 6. J.
Biochem.
ORCADIAN RHYTHMS IN RAT INTESTINAL DIGESTIVE ENZYMES
with a dark night. They became adapted to the new feeding schedule within 3 days, and their body weights increased in the same way as those of rats fed ad libitum. Figure 4 shows the enzyme activities in segment 2 of
479
rats under this daytime feeding schedule. All the enzyme activities tested were high during the daytime with the peak activities at 1600 h, which were about 2-fold higher than the lowest activities at 0400 h. Thus, when rats were fed from 0900 h to 1500 h, the phase of circadian variations shifted about half a day relative to those of rats fed ad libitum, but the ampli-' tudes of the rhythms did not change. Similar shifts in the rhythms of maltase, sucrase, and leucine aminopeptidase were also observed in segment 4. Trehalase and alkaline phosphatase in segment 4 showed no circadian variation, either when rats were fed in the daytime or fed ad libitum. DISCUSSION Disaccharidases in the small intestine are believed to be important in the final stage of digestion of disaccharides. The present results on the distribution of the activities of disaccharidases in the small intestine are in agreement with the data previously reported (13— 15). In general, disaccharidases are concentrated in the jejunum (Table I). This distribution seems to be consistent with the view that the jejunum is the region of the most active digestion and absorption of saccharides. It has been suggested that alkaline phosphatase in the small intestine also acts mainly as a digestive enzyme, though there is no direct evidence for this ( / ) . If this is so, judging from its distribution, its digestive function is greatest in the duodenum and the jejunum.
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Fig. 4. Circadian variations of enzyme activities in the small intestine of rats fed from 0900 h to 1500 h for 2 weeks. Rats were fed from 0900 h to 1500 h for 2 weeks, and were decapitated at 4 hr intervals. Enzyme activities were measured in homogenates of the mucosa of segment 2, and are expressed as ^moles of product formed per min per mg protein of the homogenate. Values are the means of those in five rats, with the standard errors. O, maltase; • , sucrase ; A, trehalase ; • , leucine aminopeptidase; x , alkaline phosphatase. Vol. 78, No. 3, 1975
In the present study, the activities of disaccharidases and alkaline phosphatase were found to exhibit clear circadian variations, especially in the jejunum. The amplitudes of the rhythms of sucrase and maltase were greater in the jejunum than in the ileum, and alkaline phosphatase and trehalase showed rhythmic variations in the jejunum, but not the ileum. Since the rhythmic changes in enzyme activities seemed to be correlated with the rhythm of food intake, and so with the digestion of nutrients, as discussed below, these results support the view that the jejunum is the region of the most active digestion of nutrients. Similar observations were reported by Shefer
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M. SAITO, E. MURAKAMI, T. NISHIDA, Y. FUJISAWA, and M. SUDA
et al. (16), who studied the circadian variation of the intestinal 3 - hydroxy - 3 - methylglutaryl-CoA reductase [EC 1.1.1.34], which is the rate-limiting enzyme of cholesterol biosynthesis. This reductase activity showed a circadian variation in both the jejunum and the ileum, and the amplitude of the rhythm was greater in the ileum than in the jejunum. The reductase activity and the rate of cholesterol synthesis from acetate are higher in the ileum than in the jejunum, and hence, the former may be the region of more active synthesis of cholesterol (17). Leucine aminopeptidase also showed a clear circadian variation, confirming previous findings. However, the amplitude of the rhythm in the jejunum was nearly the same as that in the ileum. This enzyme is also believed to take part in the final digestion of small peptides, which probably occurs mainly in the jejunum. The physiological significance of these results is not clear at present. We previously suggested that the circadian rhythms in maltase and leucine aminopeptidase in the entire small intestine are attributable to the rhythm of food intake rather than to the light-dark cycle (3). The present study confirms this in various regions of the small intestine, and furthermore, indicates that the rhythms of other digestive enzymes, such as sucrase, trehalase, and alkaline phosphatase are also correlated with the rhythm of food intake. When rats were fed during the daytime, the phases of the circadian rhythms of their enzyme activities shifted about half a day relative to those of rats fed ad libitum. A similar shift in the circadian rhythm of transport activity of L-histidine was observed in experiments on everted rat intestine (6). The phase of rhythm of intestinal cholesterol synthesis also shifted on changing the feeding time (17). These results strongly suggest that the circadian rhythms in the digestive and absorptive functions of the small intestine are closely related to the rhythm of food intake. To clarify this relationship between food intake
and enzyme rhythms further, it is necessary to measure the meal size, as done by Suda et al. (18), and to observe the movement of food along the digestive tract. Preliminary experiments along these lines are now in progress. The authors thank Miss Y. Hanayama for technical assistance during this work. REFERENCES 1. Crane, R.K. (1968)"in Handbook of Physiology, Section 6, Alimentary Canal (Code, C.F. & Heidel, W., eds.) Vol.5, pp.2535-2542, American Physiological Society, Washington 2. Ugolev, A.M. (1965) Biol. Rev. 45, 555-595 3. Saito, M. (1972) Biochim. Biophys. Ada 286, 212-215 4. Schafer, A., Hohn, P., Mika, H , & Allbach, G. (1974) Ada Histochem. 48, 301-319 5. Bunning, E. (1958) Naturwissenschaften 45, 68 6. Furuya, S. & Yugari, Y. (1971) Biochim. Biophys. Ada 241, 245-248 7. Sigdestad, C.P. & Lesher, S. (1970) Experientia 26, 1321-1322 8. Booth, C.C. (1968) in Handbook of Physiology, Section 6, Alimentary Canal (Code, C.F. & Heidel, W., eds.) Vol.3, pp. 1513-1527, American Physiological Society, Washington 9. Dahlqvist, A. (1964) Anal. Biochem. 7, 18-25 10. Goldberg, J.A. & Rutenberg, A.M. (1958) Cancer 11, 283-291 11. Forstner, G.G., Sabesin, S.M., & Isselbacher, K.J. (1968) Biochem. J. 106, 381-390 12. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) / . Biol. Chem. 193, 265-275 13. Blair, D.G.R. & Tuba, J. (1963) Canad. J. Biochem. Physiol. 41, 905-916 14. Swaminathan, N. & Radhakrishnan, A.N. (1965) Indian J. Biochem. 2, 159-163 15. Dahlqvist, A. & Thomson, D.L. (1963) / . Physiol. 167, 193-209 16. Shefer, S., Hauser, S., Lapar, V., & Mosbach, E.H. (1972) / . Lipid Res. 13, 571-573 17. Edwards, P.A., Muroya, H., & Gould, R.G. (1972) / . Lipid Res. 13, 396-401 18. Suda, M., Nagai, K., & Nakagawa, H. (1973) / . Biochem. 73, 727-738
/ . Biochem.
Circadian Rhythms in Digestive Enzymes in the Small Intestine of Rats I. Patterns of the Rhythms in Various Regions of the Small Intestine1 Masayuki SAITO, Eiko MURAKAMI, Teruo NISHIDA, Yoshiki FUJISAWA, and Masami SUDA Department of Medical Chemistry, School of Medicine, Ehime University, Shigenobu, Ehime 791-02 Received for publication, April 14, 1975
The activities of the digestive enzymes, maltase [EC 3.2.1.20], sucrase [EC 3.2.1.26], trehalase [EC 3.2.1.28], leucine aminopeptidase [EC 3.4.11.1], and alkaline phosphatase [EC 3.1.3.1] were measured in various regions of the small intestine of rats. The activities of all these enzymes were much higher in the jejunum than in the ileum, and in the distal regions of the ileum no sucrase, trehalase or alkaline phosphatase activity was detected. In the jejunum, the activities of all the enzymes tested exhibited clear circadian variations with the highest activity at 0000—0400 h and the lowest at 1200 h when the rats were fed ad libitum. In the ileum, maltase and sucrase also exhibited circadian variations, but the amplitude of the rhythm was smaller than that in the jejunum. Trehalase and alkaline phosphatase did not show any circadian variation in the ileum. Leucine aminopeptidase showed a circadian variation in the ileum with the same amplitude as in the jejunum. The phase of the circadian variations shifted about half a day when the rats were fed in the daytime, but the amplitude of the rhythm did not change.
Biochemical and histochemical studies have indicated that the epithelial cells of the small intestine bind many hydrolytic enzymes, such as maltase [EC £ 2.1.20], sucrase [EC 3.2.1.26], trehalase [EC 3.2.1.28], lactase [EC 3.2.1.23], dipeptidases, leucine aminopeptidase [EC 3.4.11.1], and alkaline phosphatase [EC 3.1.3.1]. Some of these enyzmes have been suggested to be important in the final stage of digestion or absorption of nutrients (/, 2). We reported previously that the activities 1
This study was partly supported by grants from the Yamanouchi Foundation of Metabolic Diseases. Vol. 78, No. 3, 1975
of maltase and leucine aminopeptidase in the entire small intestine of rats exhibited circadian fluctuations, and that the phase of these circadian rhythms was shifted by changing the feeding time (3). The circadian fluctuations in the activities of some hydrolases in rat small intestine were also demonstrated histochemically by Schafer et al. (4). Other investigations have shown circadian rhythmic changes in the movement of isolated intestine (5), active trransport of L-histidine (6") and the mitotic activity of the epithelium (7). These rhythms in the small intestine seem to have a close relationship to the cycle of food
475
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intake, but few data are available on their mechanisms or physiological significance. It is well-known that digestive and absorptive activities vary in different regions of the small intestine (8). For example, disaccharidase activities are highest in the jejunum and lower in the ileum in rats, and so the jejunum may be the site of the most active hydrolysis of disaccharides. This region is also likely to be the site of most active absorption of disaccharides or the monosaccharides liberated from them. Thus, it seems likely that the circadian variation in disaccharidase should be clearer in the jejunum than in the ileum. In this work, we examined the rhythmic changes in the activities of maltase, sucrase, trehalase, leucine aminopeptidase, and alkaline phosphatase in different regions of the small intestine of rats. The effect on the rhythm of altering the feeding time was also studied. EXPERIMENTAL PROCEDURE Male Wistar strain rats (150-250 g) were kept in wire-bottomed cages at 22±2° under a conventional lighting regime with a dark night. The rats were fed on laboratory chow (Oriental Yeast Co.) and allowed free access to water. In one experiment, rats were fed ad libitum, and in the other they were fed from 0900 to 1500 h. Rats were decapitated at 4 hr intervals, and their entire small intestine was rapidly removed, and divided into six segments of equal length, which were numbered serially from the duodenal region. Each segment was washed out with cold 0.9% NaCl to remove the contents. Then the mucosa was scraped off with a glass slide and homogenized with about 10 volumes of 10 mM sodium phosphate buffer (pH 7.0) using a Teflon pestled homogenizer. Disaccharidase activities were determined by a slight modification of Dahlqvist's method (9). The assay was performed in a reaction mixture containing 40 mM maltose, sucrose or trehalose, 80 mM sodium phosphate buffer (pH 7.0) and homogenates (10-50 fig protein) in a total volume of 0.5 ml at 37° for 10—30 min. The glucose liberated was estimated using specific glucose oxidase [EC
1.1.3.4] reagents (Worthington Biochemical Co.). Leucine aminopeptidase activity was measured in a reaction mixture containing 1 mM L-leucyl-j3-naphthylamide, 1 mM CoCl2, 80 mM sodium phosphate buffer (pH 7.0), and homogenates (10—50 fig protein) in a total volume of 0.5 ml. After incubation at 37° for 10 min, the reaction was terminated by adding 0.5 ml of 10% trichloroacetic acid, and the (3naphthylamine formed was measured by the method of Goldberg and Rutenberg {10). Alkaline phosphatase activity was determined under the conditions described by Forstner et al. (11). The assay was performed in a reaction mixture containing 4 mM />-nitrophenyl phosphate, 4 mM MgCl2, 80 mM sodium glycine buffer (pH 9.5), and homogenates (10—50 fig protein) in a total volume of 0.5 ml at 37° for 2-10 min. After adding 2 ml of 0.1 N NaOH, free ^-nitrophenol was determined spectrophotometrically at 400 nm. Protein was estimated by the method of Lowry et al. with bovine serum albumin as a standard (12). RESULTS The distributions of the activities of maltase, sucrase, trehalase, leucine aminopeptidase, and alkaline phosphatase were examined in the small intestines of five rats. In this experiment, the rats were fed ad libitum and killed at 2000 h. As described in the "EXPERIMENTAL PROCEDURE," the entire small intestine was divided into six equal segments, which were numbered serially from the duodenal region. Thus, segments 2 and 6 roughly correspond to the proximal part of the jejunum and the distal part of the ileum, respectively. Sucrase activity was highest in segment 3, being significantly lower in segment 1, and also in segments 4 through 6, which showed a progressive decrease in activity, as indicated in Table I. The distribution of maltase activity was essentially the same as that of sucrase activity, although it seems that there was a significant difference between the activities in segments 3 and 4. Leucine aminopeptidase activity was found to be lower in segments 1 and 6 than in segments 2, 3, 4, and 5, the latter having similar activities. The / . Biochem.
CIRCADIAN RHYTHMS IN RAT INTESTINAL DIGESTIVE ENZYMES
477
TABLE I. Distributions of enzyme activities in the small intestine. Rats were fed ad libium, and decapitated at 2000 h. The entire small intestine was divided into six equal segments, which were numbered serially from the duodenal region. Values are the means of those in five rats, with the standard errors. Enzyme activity (/umoles of product formed per min per mg protein) Segment No. 1 2 3 4 5 6 a
Maltase
Sucrase
Trehalase
Alkaline phosphatase
Leucine aminopeptidase
0.804+0.047 0.911 ±0.035 1.10 ±0.07 1.08 ±0.03 0.698 ±0.017 0.224+0.013
0.078 ±0.002 0.130 ±0. 007 0.142 ±0.013 0.098+0.004 0.031 ±0.003 N.D."
0.114+0.001 0.093+0.003 0.059+0. 006 0.029 ±0.004 0.006 ±0.001 N.D.
1.05 +0.09 0.471+0.027 0.197+0.028 0.071+0.028 0.023+0.015 N.D.
0.053 ±0.003 0.086 ±0.003 0.089 ±0.006 0.097 ±0.006 0. 087 ±0.004 0.050 ±0.003
Not Detectable.
distribution of trehalase activity was essentially the same as that of alkaline phosphatase activity, being different from those of sucrase, maltase, and leucine aminopeptidase. The activities of trehalase and alkaline phosphatase were both highest in segment 1, and decreased progressively in segments 2 through 5, no activity being detectable in segment 6. In Table I, the enzyme activities are expressed as specific activities, i.e., ^moles of product formed per min per mg protein, and the total activity per segment is roughly proportional to the specific activity, since the protein content per segment was found to be nearly the same among segments. To examine the circadian variations of the enzyme activities in various regions of the small intestine, rats were fed ad libitum and killed at 4 hr intervals, and their enzyme activities were measured in segments 2, 4, and 6. As shown inFig. 1, maltase_ activity exhibited a clear circadian variation in all three segments. Activity was highest at 0000 h and lowest at 1200 h in all segments. The ratio of the highest to the lowest activity, i.e. the amplitude of the rhythm, was about 2.6 in segment 2, which was significantly higher than those in segments 4 and 6 (1.8 and 1.7, respectively). Sucrase activity also showed a circadian fluctuation in segments 2 and 4, with the highest activity at 0000 h and the lowest at 1200 h, and the ratio of the former to the Vol. 78, No. 3, 1975
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Fig. 1. Circadian variations of mallase activity in various regions of the small intestine. Rats were fed ad libitum, and decapitated at 4 hr intervals. Segments of the small intestine were obtained as for Table I, and maltase activity was measured in homogenates of the mucosa of each segment. Values are the means of those in five rats, with the standard errors. O, segment 2; 0 , segment 4; x , segment 6.
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M. SAITO, E. MURAKAMI, T. NISHIDA, Y. FUJISAWA, and M. SUDA
latter was 2.0 in segment 2, which was definitely higher than that of 1.5 in segment 4 (data not shown). In segment 6, no activity was detected at any time of the day. Figure 2 shows the circadian fluctuations of alkaline phosphatase in segments 2 and 4. In segment 2, the activity exhibited a clear circadian variation with the same pattern as those of maltase and sucrase, and the amplitude of the rhythm was 2.1. However, in segment 4, the activity showed no circadian variation. Segment 4 also showed no circadian variation in trehalase activity, which exhibited a clear circadian variation in segment 2 with the same pattern as those of alkaline phosphatase, maltase, and sucrase. In segment 6, no activity of alkaline phosphatase or trehalase was detected at any time of the day. Leucine aminopeptidase activity also showed a clear circadian variation with the highest activity at 0400 h in all segments examined, as shown in Fig. 3. However, there was no significant difference in the amplitudes of the rhythms in segments 2, 4, and 6, unlike in
the cases of maltase, sucrase, alkaline phosphatase, and trehalase. In summary, all the enzyme activities tested exhibited clear circadian fluctuations in the small intestine with the highest activities 0000-0400 h and the lowest at 1200 h. The amplitudes of the rhythms of disaccharidases and alkaline phosphatase were highest in segment 2, corresponding to the proximal region of the jejunum, and the rhythmic variation was less marked or not detected in segments 4 and 6, corresponding to the middle region of the small intestine and the distal region of the ileum, respectively. Rats are nocturnal, so they eat during the night when they are fed ad libitum. As described above, the enzyme activities in the intestine were found to be higher at night. Next, the relationship between the rhythms of the enzyme activities and food intake was examined. Rats were fed during the daytime from 0900 h to 1500 h every day for 2 weeks, and kept under a conventional lighting schedule
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Fig. 2. Circadian variations of alkaline phosphatase activity in various regions of the small intestine. Alkaline phosphatase activity was measured in the same homogenate used for Fig. 1. Values are the means of those in five rats, with the standard errors. O, segment 2; • , segment 4.
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Fig. 3. Circadian variations of leucine aminopeptidase activity in various regions of the small intestine. Leucine aminopeptidase activity was measured in the same homogenate used for Fig. 1. Values are the means of those in five rats, with the standard errors. O, segment 2; • , segment 4; x, segment 6. J.
Biochem.
ORCADIAN RHYTHMS IN RAT INTESTINAL DIGESTIVE ENZYMES
with a dark night. They became adapted to the new feeding schedule within 3 days, and their body weights increased in the same way as those of rats fed ad libitum. Figure 4 shows the enzyme activities in segment 2 of
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rats under this daytime feeding schedule. All the enzyme activities tested were high during the daytime with the peak activities at 1600 h, which were about 2-fold higher than the lowest activities at 0400 h. Thus, when rats were fed from 0900 h to 1500 h, the phase of circadian variations shifted about half a day relative to those of rats fed ad libitum, but the ampli-' tudes of the rhythms did not change. Similar shifts in the rhythms of maltase, sucrase, and leucine aminopeptidase were also observed in segment 4. Trehalase and alkaline phosphatase in segment 4 showed no circadian variation, either when rats were fed in the daytime or fed ad libitum. DISCUSSION Disaccharidases in the small intestine are believed to be important in the final stage of digestion of disaccharides. The present results on the distribution of the activities of disaccharidases in the small intestine are in agreement with the data previously reported (13— 15). In general, disaccharidases are concentrated in the jejunum (Table I). This distribution seems to be consistent with the view that the jejunum is the region of the most active digestion and absorption of saccharides. It has been suggested that alkaline phosphatase in the small intestine also acts mainly as a digestive enzyme, though there is no direct evidence for this ( / ) . If this is so, judging from its distribution, its digestive function is greatest in the duodenum and the jejunum.
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Fig. 4. Circadian variations of enzyme activities in the small intestine of rats fed from 0900 h to 1500 h for 2 weeks. Rats were fed from 0900 h to 1500 h for 2 weeks, and were decapitated at 4 hr intervals. Enzyme activities were measured in homogenates of the mucosa of segment 2, and are expressed as ^moles of product formed per min per mg protein of the homogenate. Values are the means of those in five rats, with the standard errors. O, maltase; • , sucrase ; A, trehalase ; • , leucine aminopeptidase; x , alkaline phosphatase. Vol. 78, No. 3, 1975
In the present study, the activities of disaccharidases and alkaline phosphatase were found to exhibit clear circadian variations, especially in the jejunum. The amplitudes of the rhythms of sucrase and maltase were greater in the jejunum than in the ileum, and alkaline phosphatase and trehalase showed rhythmic variations in the jejunum, but not the ileum. Since the rhythmic changes in enzyme activities seemed to be correlated with the rhythm of food intake, and so with the digestion of nutrients, as discussed below, these results support the view that the jejunum is the region of the most active digestion of nutrients. Similar observations were reported by Shefer
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M. SAITO, E. MURAKAMI, T. NISHIDA, Y. FUJISAWA, and M. SUDA
et al. (16), who studied the circadian variation of the intestinal 3 - hydroxy - 3 - methylglutaryl-CoA reductase [EC 1.1.1.34], which is the rate-limiting enzyme of cholesterol biosynthesis. This reductase activity showed a circadian variation in both the jejunum and the ileum, and the amplitude of the rhythm was greater in the ileum than in the jejunum. The reductase activity and the rate of cholesterol synthesis from acetate are higher in the ileum than in the jejunum, and hence, the former may be the region of more active synthesis of cholesterol (17). Leucine aminopeptidase also showed a clear circadian variation, confirming previous findings. However, the amplitude of the rhythm in the jejunum was nearly the same as that in the ileum. This enzyme is also believed to take part in the final digestion of small peptides, which probably occurs mainly in the jejunum. The physiological significance of these results is not clear at present. We previously suggested that the circadian rhythms in maltase and leucine aminopeptidase in the entire small intestine are attributable to the rhythm of food intake rather than to the light-dark cycle (3). The present study confirms this in various regions of the small intestine, and furthermore, indicates that the rhythms of other digestive enzymes, such as sucrase, trehalase, and alkaline phosphatase are also correlated with the rhythm of food intake. When rats were fed during the daytime, the phases of the circadian rhythms of their enzyme activities shifted about half a day relative to those of rats fed ad libitum. A similar shift in the circadian rhythm of transport activity of L-histidine was observed in experiments on everted rat intestine (6). The phase of rhythm of intestinal cholesterol synthesis also shifted on changing the feeding time (17). These results strongly suggest that the circadian rhythms in the digestive and absorptive functions of the small intestine are closely related to the rhythm of food intake. To clarify this relationship between food intake
and enzyme rhythms further, it is necessary to measure the meal size, as done by Suda et al. (18), and to observe the movement of food along the digestive tract. Preliminary experiments along these lines are now in progress. The authors thank Miss Y. Hanayama for technical assistance during this work. REFERENCES 1. Crane, R.K. (1968)"in Handbook of Physiology, Section 6, Alimentary Canal (Code, C.F. & Heidel, W., eds.) Vol.5, pp.2535-2542, American Physiological Society, Washington 2. Ugolev, A.M. (1965) Biol. Rev. 45, 555-595 3. Saito, M. (1972) Biochim. Biophys. Ada 286, 212-215 4. Schafer, A., Hohn, P., Mika, H , & Allbach, G. (1974) Ada Histochem. 48, 301-319 5. Bunning, E. (1958) Naturwissenschaften 45, 68 6. Furuya, S. & Yugari, Y. (1971) Biochim. Biophys. Ada 241, 245-248 7. Sigdestad, C.P. & Lesher, S. (1970) Experientia 26, 1321-1322 8. Booth, C.C. (1968) in Handbook of Physiology, Section 6, Alimentary Canal (Code, C.F. & Heidel, W., eds.) Vol.3, pp. 1513-1527, American Physiological Society, Washington 9. Dahlqvist, A. (1964) Anal. Biochem. 7, 18-25 10. Goldberg, J.A. & Rutenberg, A.M. (1958) Cancer 11, 283-291 11. Forstner, G.G., Sabesin, S.M., & Isselbacher, K.J. (1968) Biochem. J. 106, 381-390 12. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) / . Biol. Chem. 193, 265-275 13. Blair, D.G.R. & Tuba, J. (1963) Canad. J. Biochem. Physiol. 41, 905-916 14. Swaminathan, N. & Radhakrishnan, A.N. (1965) Indian J. Biochem. 2, 159-163 15. Dahlqvist, A. & Thomson, D.L. (1963) / . Physiol. 167, 193-209 16. Shefer, S., Hauser, S., Lapar, V., & Mosbach, E.H. (1972) / . Lipid Res. 13, 571-573 17. Edwards, P.A., Muroya, H., & Gould, R.G. (1972) / . Lipid Res. 13, 396-401 18. Suda, M., Nagai, K., & Nakagawa, H. (1973) / . Biochem. 73, 727-738
/ . Biochem.
