DEVELOPMENTAL
BIOLOGY
60, 487-492 (1977)
Lack of Circadian
NANCY Department
Rhythmicity of Rat Fetal Intestinal Compared to the Dams
R. STEVENSON,’
of Physiology,
M. BYRA,
WILLIAM
College of Medicine Piscatuway,
AND
SUSAN
Enzymes
E. DAY
and Dentistry of New Jersey, Rutgers Medical New Jersey 08854
Received May 19,1977;
as
School,
accepted June 29,1977
The 24-hr activity patterns of intestinal maltase, lactase, leucylnaphthylamine hydrolyzing activity, y-glutamyltransferase, and alkaline phosphatase were determined in pregnant rats maintained on a 12-12 light-dark cycle, with feeding during the dark period (1800-0600 hr, EST). The activities of these enzymes plus those of lysosomal maltase and lactase were followed during the same time period in 19- to 20-day-old fetuses. The activity patterns in the dams followed circadian rhythms, with peak activities occurring during the feeding-dark period. These rhythms are similar to the feeding schedule-cued rhythms observed in male rats and, therefore, are assumed to be feeding schedule cued also. In the fetuses, which obtained nutrients through the placenta, the activities increased in a somewhat nonlinear manner throughout the entire 24-hr period, but did not display a defined rhythm. It is concluded that endogenous intestinal enzyme rhythms do not exist in utero, and that oral and/or intermittent feeding is necessary for these rhythms to occur. INTRODUCTION
Circadian rhythms cued by feeding schedule have been observed in the activity patterns of a number of intestinal brush border digestive and absorptive functions in the adult male rat (Furuya and Yugari, 1971, 1974; Saito, 1972; Saito et al., 1976; Stevenson et al., 1975; Stevenson and Fierstein, 1976). These functions include the enzymes sucrase, maltase, lactase, trehalase, leucylnaphthylamine hydrolyzing activity, y-glutamyltransferase, alkaline phosphatase and the transport systems for monosaccharides and L-histidine. When adult rats were subjected to starvation, the rhythms observed in the activity of sucrase and maltase continued for 2 days and were essentially extinguished by the fifth day (Saito et al., 1976). Similar fadeout has been suggested to occur in the rhythm patterns of L-histidine and n-glu’ To whom correspondence
should be addressed.
case transport (Furuya and Yugari, 1971, 1974). When rats were infused intravenously with a liquid diet for 7 days, the rhythms of most but not all of the abovementioned enzymes were extinguished (Sitren and Stevenson, 1977). Thus, even with the maintenance of the nutritional status, the existence of most of these rhythms in the adult cannot be maintained in the absence of oral and/or intermittent nutrient intake. In general, little work has been done on enzyme rhythms in fetal development. A number of digestive and absorptive functions are present in the fetal intestine (Deren, 1968), although their use at that time is not essential for fetal nutrition. The nutritional needs are provided via the blood stream at a more or less constant rate. Therefore, the fetus presents a physiological situation in which to study the existence of rhythmicity in digestive enzymes removed from external cues. The present study was designed to deter-
487 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0012-1606
488
DEVELOPMENTAL
BIOLOGY
mine the existence of endogenous rhythms in fetal intestinal enzymes and the need of oral and/or intermittent intake for the maintenance of these rhythms. The enzyme systems studied in both the fetus and dam were: lactase (EC 3.2.1.231, y-glutamyltransferase (EC 2.3.2.21, alkaline phosphoatase (EC 3.1.3.11, (EC 3.2.1.201, and leucylnaphthylamide hydrolyzing activity. We have also studied fetal acid lactase and maltase which are presumably of lysosomal origin. These latter two enzymes have been shown to be present in significant amounts in the fetal intestine (Doe11 and Kretchmer, 1962; Galand and Forstner, 1974). METHODS
Animals. Pregnant Sprague-Dawley rats were obtained from the Camm Research Institute. Breeding occurred at night, between 1800 and 0900 hr Eastern Standard Time (EST); the next day was considered Day 1 of gestation. Animals were received on the thirteenth day of gestation and were housed in a constant-environment room. They were caged individually in plastic cages with ground corn cobs for bedding and shredded paper for nesting material. A 12-12-hr light-dark cycle, with darkness from 1800 to 0600 hr EST, was used. Purina Rat Chow was available only during the 12 hr of darkness, and water was available ad libitum. The animals were not handled after the initial weigh-in until a final weight was obtained prior to killing. Food was added or removed from the cages during the 10 min preceding or following the feeding period. Starting at 1700 hr EST on the nineteenth day of gestation, groups of two to four rats were killed by cervical dislocation every 4 hr, for the next 24 hr; that is, at 1700, 2100, 0100, 0500, 0900, 1300, and 1700 hr EST. An extra 5 to 10 pregnant rats were obtained and maintained with the experimental animals, and those animals with
VOLUME
60, 1977
the highest daily food intake were used in the experiment. Unused pregnant rats all delivered litters within 36 hr of the end of the experiment. Procedures. The entire small intestine of the dam was removed, rinsed in cold 0.9% saline, and everted, and the mucosa was scraped. The scrapings were homogenized in saline, and aliquots were frozen at -20°C for later assay of protein and enzymes. The uterus was removed and placed in ice-cold saline. Then the fetuses were removed, rinsed in fresh saline, blotted, and weighed. The fetal viscera were removed from the body and were placed on an iced plate with a small amount of cold saline. The small intestine, from the stomach to the ileocecal junction, was dissected free, and its length was measured. All of the fetal intestines from each litter were pooled and homogenized in cold saline. Aliquots of the homogenate were frozen for later assay of enzymes and protein. The disaccharidases were assayed according to the Lloyd and Whelan (1969) modification of the Dahlqvist (1964) method; alkaline phosphatase was assayed by the method of Eichholz (1967), y-glutamyltransferase, by the method of Naftalin et al. (1969); leucylnaphthylamine hydrolyzing activity, by the method of Goldbarg and Rutenberg (1958); acid maltase and lactase by the method of Galand and Forstner (1974); and protein by the method of Lowry et al. (1951) using crystalline bovine serum albumin as standard. Normal fetal growth and litter size were assessed by comparison to the data of Golzalez (1932) and Villee and Hagerman (1958). Data calculations and statistical evaluation. The homogenates of each dam and litter were assayed separately. Enzyme and protein assays were done in quadruplicate. Mean individual dam or litter values were used in the calculations. Enzyme activity of the dams was expressed as specific activities, micromoles of substrate hydrolyzed per minute per milligram of protein. The fetal enzyme activities were ex-
489
BRIEF NOTES
pressed as micromoles of substrate hydrolyzed per minute per centimeter intestinal length. Fetal specific activities, micromoles of substrate hydrolyzed per minute per milligram of protein, were also calculated and used in analyzing the data, but are not shown. The data represent the means f SE of four to six dams or litters, obtained from replicate experiments, at each time point. Least-squares analysis was done to assess linearity of the data. The resulting straight-line functions were superimposed on the fetal curves. Slopes, B, and coefficients of determination, +, were obtained from this analysis.
o-rat
t
feedtng and dark
070 062 046
‘0050
038
-0046 -0042
030 054 I t
-0038
0011
-0034
0010
-0030 -0.026
G e 0009
-0022 I
s 0007
g :$ :
2
?
0006
;
0005
004s ;
000
0.044
0003 I =; 0008
0 040 t
0 036 0 032
052 046
RESULTS
044 04
Specific enzyme activities in the dams 03 032 followed circadian rhythm patterns (Fig. 056 I, 1 , , , ,700 2100 0100 0500 0900 1300 1700 1). Total protein content of the mucosal Time (EST1 homogenates did not change during the 24FIG. 1. Rhythmicity in the specific activities of hr period. As observed in previous work, alkaline phosphatase CAP), y-glutamyltransferase alkaline phosphatase activity did not re- (yGT), lactase (lact), leucylnaphthylamine hydroturn to the initial level 24 hr later. Peak lyzing activity (LNAase), and maltase (malt) in activities were observed during the feed- dams. Mean 5 SE (n = 4-6). ing-dark period. Maltase reached maximum activity prior to the other enzymes, Visual inspection of this data shows the and alkaline phosphatase peaked after the change in length occurring in a start-stop fashion, with the suggestion of a cyclic other enzymes. Fetal body weight (Fig. 2) and the num- pattern repeating every 8 hr. In contrast to the enzyme activities of ber of fetuses per litter (12 * 3, n = 41, mean * SD) were similar to those observed the dams, the fetal enzyme activities did in litters from ad libitum-fed dams. Rapid not follow circadian rhythm patterns fetal growth during the time course of the (Figs. 2, 3). These enzyme activities did experiment was demonstrated by the in- increase during the 24-hr period. The increases in fetal body weight, total small creases appeared to be related closely to intestinal protein content, and small intes- the increases in the length of the small tinal length (Fig. 2). Fetal body weight intestine and somewhat less closely to inincreased linearly with time, B = 0.07, r2 creases in total intestinal protein content. = 83%. The total small intestinal protein Both sets of data versus time were subcontent, B = 0.10, and length, B = 0.11, jected to least-squares analysis for fitting increased somewhat more rapidly than did to a straight line. The resulting coeffithe body weight during this period. The cients of determination are shown in Table protein content increased in a linear man- 1. The enzyme activities, when related to ner, r2 = 82%. On the other hand, the length of the small intestine, were better elongation of the small intestine was not described by a straight-line function than well described by a linear function, r2 = when related to protein content, except for 47%. Less than 50% of the change in length lactase activity at pH 6, which was decan be attributed to a steady growth rate. scribed equally well by both relationships.
490
DEVELOPMENTAL
90r
1
BIOLOGY
VOLUME
60. 1977 DISCUSSION
feeding and dark
In the dams the activities of certain brush border enzymes, maltase, lactase, -100 - 92
-
6.4: 7.62 66~ 6.0: 524; - 4.4 2 - 3.6w 2.6 >20 f
7.0r 70. 6.2 5.4. 4.6 . 3.6. 3.0. %I 2.2 . 5
Mdl
5 4 3 2
I
1.2 ” -10: - 0.0 = - 0.6 5 - 0.4 ; -0.2 4 -0.0 z
.’
, , , , 1700 2100 0100 0500 0900 Time
, , 1300 1700
(EST)
2. Changes in leucylnaphthylamine hydrolyzing activity (LNAase), alkaline phosphatase (AP), small intestinal length (Sm. I. Ln.), small intestinal protein (Protein), and body weight (Body Wt.) in 19- to ZO-day-old fetuses. Mean 2 SE (n = 46). Least-squares determined line (- - -1. FIG.
The extremely low rz value of maltase activity at pH 3 was presumably due to the variability between litters at 0900 and 1300 hr. Coefficients of variation at these times were 42 and 44%, respectively. All of the fetal brush border enzyme activities appeared to increase at a considerably greater rate than did elongation of the small intestine (Figs. 2, 3). The brush border enzymes also increased in activity more rapidly than the intracellular lysosoma1 enzymes. In the graphs of the two disaccharidases, the activities at different pHs are graphed to the same scale in order to show the differences in slopes (Fig. 3). Considering that some of the enzyme activity at pH 6 is due to the presence of the lysosomal enzymes, the activity of the brush border enzymes would be increasing at an even greater rate than indicated.
Time
(EST1
3. Changes in y-glutamyltransferase (yGT), maltase (malt) activity measured at pH 6 and 3, and lactase (lact) activity measured at pH 6 and 3, in 19to 20-day-old fetuses. Mean + SE (n = 4-61. Leastsquares determined line (- - -). FIG.
TABLE VARIATION ACCOUNTED
1
IN FETAL ENZYME ACTIVITIES FOR BY THE FITTED REGRESSION
Coefficient of determination (r2)
Fetal enzyme
Leneth” Leucylnaphthylamine drolyzing activity Alkaline phosphatase y-Glutamyltransferase Lactase PH 6 PH 3 Maltase PH 6 DH 3
hy-
a Micromoles of substrate per length versus time. b Micromoles of substrate per protein versus time.
Proteinb
a7
69
79 72
64 32
79 77
78 61
76 63
56 17
hydrolyzed
per minute
hydrolyzed
per minute
491
BRIEF NOTES
leucylnaphthylamine hydrolyzing activity, and y-glutamyltransferase, follow circadian rhythms similar to those seen in male rats (Saito, 1972; Saito et al., 1976; Stevenson and Fierstein, 1976; Stevenson et al., 1975). The rhythm of alkaline phosphatase activity follows a slightly different periodicity. This is also seen in ad libitumfed male rats (Stevenson et al., 1975). In light of these similarities in enzyme activity patterns, it is assumed that, as in the male, these rhythms in the pregnant rat are cued chiefly or exclusively by the feeding schedule. In contrast, the activity patterns of these enzymes plus those of lysosomal maltase and lactase do not follow a circadian pattern in the 19- to 20-day-old fetuses. The possibility of endogenous rhythms of low amplitude and shortened cycle in these fetal enzymes is intriguing, but from this work it seems unlikely. During this stage of development the small intestine does not mature as a unified structure, but proliferation and differentiation of the villus surface occur at different times in different parts of the intestine (Kammeraad, 1942; Lindberg and Owman, 1966). It is reasonable to consider that the brush border enzymes of the absorptive cell would also be synthesized at different rates in the various areas of the intestine, and, thus, the nonlinear variation in enzyme activities of the whole intestine is a reflection of these differences. Presumably, the same processes cause the nonlinear variation in the lysosomal enzymes and in the elongation of the small intestine. Therefore, it may be fortuitous that the summation of the various processes in the epithelial cell surface and underlying cell layers results in a steady increase in total small intestinal protein content. By relating enzyme activity to intestinal length it was hoped to compensate for changes due to the general maturation processes and to leave only variability in
the enzyme activity. This additional variation is considered to result from random scatter and not from an endogenous rhythm. It is concluded that endogenous rhythms do not exist in the activity patterns of these brush border enzymes prior to birth. This observation coupled with the starvation and intravenous feeding studies done with adult rats leads to the further conclusion that intermittent and/or oral feeding not only cues these rhythms but is also involved in maintaining them. Since circadian rhythms are not present in fetal enzyme activities and do occur in the adult enzyme patterns, one may speculate about when these rhythms appear. If intermittent oral feeding is the only factor required to initiate these rhythms, then they would be expected to appear very soon after birth; by 6 to 6 days neonates suckle predominantly during the day (Walker et al., 1974). If other factors are involved, such as the changes in small intestinal structure and enzyme profile and the blood hormone levels which occur about the time of weaning (Deren, 1968), then circadian rhythmicity would not be apparent until after weaning. We thank Mrs. Lisa Wolff and Mr. Scot Zeigen for their technical assistance. This work was supported in part by Grant AM 18953 from the National Institute of Arthritis, Metabolic and Digestive ,Diseases and by a GRS-MSRP grant. REFERENCES DAHLQVIST, A. (1964). Method for assay of intestinal disaccharidases. Anal. Biochem. 7, 18-25. DEREN, J. J. (1968). Development of intestinal structure and function. In “Handbook of Physiology” (C. F. Code, ed.), Vol. 3, pp. 1099-1123. American Physiological Society, Washington, D.C. DOELL, R. G., and KRETCHMER, N. (1962). Studies on small intestine during development. I. Distribution and activity of P-galactosidase. Biochim. Biophys. Acta 62, 353-362. EICHHOLZ, A. (1967). Structural and functional organization of the brush border of intestinal epithelial cells. III. Enzymic activities and chemical composition of various fractions of Tris-disrupted
492
DEVELOPMENTAL BIOLOGY
brush borders. Biochim. Biophys. Actu 135, 475 482. FURUYA, S., and YUGARI, Y. (1971). Daily rhythmic change in the transport of histidine by everted sacs of rat small intestine. Biochim. Biophys. Actu 241, 245-248. FURUYA, S., and YUGARI, Y. (1974). Daily rhythmic change of L-histidine and glucose absorptions in rat small intestine in uiuo. Biochim. Biophys. Actu 343, 558-564. GALAND, G., and FORSTNER, G. G. (1974). Soluble neutral and acid maltases in the suckling-rat intestine. Biochem. J. 144, 281-292. GOLDBARG, J. A., and RUTENBERG, A. M. (1958). The calorimetric determination of leucine aminopeptidase in urine and serum of normal subjects and patients with cancer and other diseases. Cancer 1, 283-288. GOLZALEZ, A. W. A. (1932). The prenatal growth of the albino rat. And. Rec. 52, 117-138. KAMMERAAD, A. (1942). The development of the gastro-intestinal tract of the rat. J. Morphol. 70,323342. LINDBERG, T., and OWMAN, C. (1966). Intestinal dipeptidase. Development of dipeptidase activity in the small intestine of the rat as related to the development of the intestinal mucosa. Actu Physiol. Scam!. 68, 141-151. LLOYD, J. B., and WHELAN, W. J. (1969). An improved method for enzymic determination of glucose in the presence of maltose. Anal. Biochem. 30, 467-469. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurements
VOLUME 60, 1977
with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. NAFTALIN, L., SEXTON, M., WHITAKER, J. F., and TRACEY, D. (19691. A routine procedure for estimating serum y-glutamyltranspeptidase activity. Clin. Chim. Actu 26, 293-296. SAITO, M. (1972). Daily rhythmic changes in brush border enzymes of the small intestine and kidney in rat. Biochim. Biophys. Acta 286, 212-215. SAITO, M., MURAKAMI, E., and SUDA, M. (1976). Circadian rhythms in disaccharidases of rat small intestine and its relation to food intake. Biochim. Biophys. Actu 421, 177-179. SITREN, H., and STEVENSON, N. R. (1977). Rhythmic fluctuations in small intestinal functions, insulin, and gastrin in orally and intravenously fed rats. Gustroenterology 72, 1133. STEVENSON, N. R., and FIERSTEIN, J. S. (1976). Circadian rhythms of intestinal sucrase and glucose transport: cued by time of feeding. Amer. J. Physiol. 230, 731-735. STEVENSON, N. R., FERRIGNI, F., PARNICKY, K., DAY, S., and FIERSTEIN, J. S. (1975). Effect of changes in feeding schedule on the diurnal rhythms and daily activity levels of intestinal brush border enzymes and transport systems. Biochim. Biophys. Actu 406, 131-145. VILLEE, C. A., and HAGERMAN, D. D. (1958). Effect of oxygen deprivation on the metabolism of fetal and adult tissues. Amer. J. Physiol. 194, 457-464. WALKER, P. R., BONNEY, R. J., and POTTER, W. R. (1974). Diurnal rhythms of hepatic carbohydrate metabolism during development of the rat. Biothem. J. 140, 523-529.
BIOLOGY
60, 487-492 (1977)
Lack of Circadian
NANCY Department
Rhythmicity of Rat Fetal Intestinal Compared to the Dams
R. STEVENSON,’
of Physiology,
M. BYRA,
WILLIAM
College of Medicine Piscatuway,
AND
SUSAN
Enzymes
E. DAY
and Dentistry of New Jersey, Rutgers Medical New Jersey 08854
Received May 19,1977;
as
School,
accepted June 29,1977
The 24-hr activity patterns of intestinal maltase, lactase, leucylnaphthylamine hydrolyzing activity, y-glutamyltransferase, and alkaline phosphatase were determined in pregnant rats maintained on a 12-12 light-dark cycle, with feeding during the dark period (1800-0600 hr, EST). The activities of these enzymes plus those of lysosomal maltase and lactase were followed during the same time period in 19- to 20-day-old fetuses. The activity patterns in the dams followed circadian rhythms, with peak activities occurring during the feeding-dark period. These rhythms are similar to the feeding schedule-cued rhythms observed in male rats and, therefore, are assumed to be feeding schedule cued also. In the fetuses, which obtained nutrients through the placenta, the activities increased in a somewhat nonlinear manner throughout the entire 24-hr period, but did not display a defined rhythm. It is concluded that endogenous intestinal enzyme rhythms do not exist in utero, and that oral and/or intermittent feeding is necessary for these rhythms to occur. INTRODUCTION
Circadian rhythms cued by feeding schedule have been observed in the activity patterns of a number of intestinal brush border digestive and absorptive functions in the adult male rat (Furuya and Yugari, 1971, 1974; Saito, 1972; Saito et al., 1976; Stevenson et al., 1975; Stevenson and Fierstein, 1976). These functions include the enzymes sucrase, maltase, lactase, trehalase, leucylnaphthylamine hydrolyzing activity, y-glutamyltransferase, alkaline phosphatase and the transport systems for monosaccharides and L-histidine. When adult rats were subjected to starvation, the rhythms observed in the activity of sucrase and maltase continued for 2 days and were essentially extinguished by the fifth day (Saito et al., 1976). Similar fadeout has been suggested to occur in the rhythm patterns of L-histidine and n-glu’ To whom correspondence
should be addressed.
case transport (Furuya and Yugari, 1971, 1974). When rats were infused intravenously with a liquid diet for 7 days, the rhythms of most but not all of the abovementioned enzymes were extinguished (Sitren and Stevenson, 1977). Thus, even with the maintenance of the nutritional status, the existence of most of these rhythms in the adult cannot be maintained in the absence of oral and/or intermittent nutrient intake. In general, little work has been done on enzyme rhythms in fetal development. A number of digestive and absorptive functions are present in the fetal intestine (Deren, 1968), although their use at that time is not essential for fetal nutrition. The nutritional needs are provided via the blood stream at a more or less constant rate. Therefore, the fetus presents a physiological situation in which to study the existence of rhythmicity in digestive enzymes removed from external cues. The present study was designed to deter-
487 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0012-1606
488
DEVELOPMENTAL
BIOLOGY
mine the existence of endogenous rhythms in fetal intestinal enzymes and the need of oral and/or intermittent intake for the maintenance of these rhythms. The enzyme systems studied in both the fetus and dam were: lactase (EC 3.2.1.231, y-glutamyltransferase (EC 2.3.2.21, alkaline phosphoatase (EC 3.1.3.11, (EC 3.2.1.201, and leucylnaphthylamide hydrolyzing activity. We have also studied fetal acid lactase and maltase which are presumably of lysosomal origin. These latter two enzymes have been shown to be present in significant amounts in the fetal intestine (Doe11 and Kretchmer, 1962; Galand and Forstner, 1974). METHODS
Animals. Pregnant Sprague-Dawley rats were obtained from the Camm Research Institute. Breeding occurred at night, between 1800 and 0900 hr Eastern Standard Time (EST); the next day was considered Day 1 of gestation. Animals were received on the thirteenth day of gestation and were housed in a constant-environment room. They were caged individually in plastic cages with ground corn cobs for bedding and shredded paper for nesting material. A 12-12-hr light-dark cycle, with darkness from 1800 to 0600 hr EST, was used. Purina Rat Chow was available only during the 12 hr of darkness, and water was available ad libitum. The animals were not handled after the initial weigh-in until a final weight was obtained prior to killing. Food was added or removed from the cages during the 10 min preceding or following the feeding period. Starting at 1700 hr EST on the nineteenth day of gestation, groups of two to four rats were killed by cervical dislocation every 4 hr, for the next 24 hr; that is, at 1700, 2100, 0100, 0500, 0900, 1300, and 1700 hr EST. An extra 5 to 10 pregnant rats were obtained and maintained with the experimental animals, and those animals with
VOLUME
60, 1977
the highest daily food intake were used in the experiment. Unused pregnant rats all delivered litters within 36 hr of the end of the experiment. Procedures. The entire small intestine of the dam was removed, rinsed in cold 0.9% saline, and everted, and the mucosa was scraped. The scrapings were homogenized in saline, and aliquots were frozen at -20°C for later assay of protein and enzymes. The uterus was removed and placed in ice-cold saline. Then the fetuses were removed, rinsed in fresh saline, blotted, and weighed. The fetal viscera were removed from the body and were placed on an iced plate with a small amount of cold saline. The small intestine, from the stomach to the ileocecal junction, was dissected free, and its length was measured. All of the fetal intestines from each litter were pooled and homogenized in cold saline. Aliquots of the homogenate were frozen for later assay of enzymes and protein. The disaccharidases were assayed according to the Lloyd and Whelan (1969) modification of the Dahlqvist (1964) method; alkaline phosphatase was assayed by the method of Eichholz (1967), y-glutamyltransferase, by the method of Naftalin et al. (1969); leucylnaphthylamine hydrolyzing activity, by the method of Goldbarg and Rutenberg (1958); acid maltase and lactase by the method of Galand and Forstner (1974); and protein by the method of Lowry et al. (1951) using crystalline bovine serum albumin as standard. Normal fetal growth and litter size were assessed by comparison to the data of Golzalez (1932) and Villee and Hagerman (1958). Data calculations and statistical evaluation. The homogenates of each dam and litter were assayed separately. Enzyme and protein assays were done in quadruplicate. Mean individual dam or litter values were used in the calculations. Enzyme activity of the dams was expressed as specific activities, micromoles of substrate hydrolyzed per minute per milligram of protein. The fetal enzyme activities were ex-
489
BRIEF NOTES
pressed as micromoles of substrate hydrolyzed per minute per centimeter intestinal length. Fetal specific activities, micromoles of substrate hydrolyzed per minute per milligram of protein, were also calculated and used in analyzing the data, but are not shown. The data represent the means f SE of four to six dams or litters, obtained from replicate experiments, at each time point. Least-squares analysis was done to assess linearity of the data. The resulting straight-line functions were superimposed on the fetal curves. Slopes, B, and coefficients of determination, +, were obtained from this analysis.
o-rat
t
feedtng and dark
070 062 046
‘0050
038
-0046 -0042
030 054 I t
-0038
0011
-0034
0010
-0030 -0.026
G e 0009
-0022 I
s 0007
g :$ :
2
?
0006
;
0005
004s ;
000
0.044
0003 I =; 0008
0 040 t
0 036 0 032
052 046
RESULTS
044 04
Specific enzyme activities in the dams 03 032 followed circadian rhythm patterns (Fig. 056 I, 1 , , , ,700 2100 0100 0500 0900 1300 1700 1). Total protein content of the mucosal Time (EST1 homogenates did not change during the 24FIG. 1. Rhythmicity in the specific activities of hr period. As observed in previous work, alkaline phosphatase CAP), y-glutamyltransferase alkaline phosphatase activity did not re- (yGT), lactase (lact), leucylnaphthylamine hydroturn to the initial level 24 hr later. Peak lyzing activity (LNAase), and maltase (malt) in activities were observed during the feed- dams. Mean 5 SE (n = 4-6). ing-dark period. Maltase reached maximum activity prior to the other enzymes, Visual inspection of this data shows the and alkaline phosphatase peaked after the change in length occurring in a start-stop fashion, with the suggestion of a cyclic other enzymes. Fetal body weight (Fig. 2) and the num- pattern repeating every 8 hr. In contrast to the enzyme activities of ber of fetuses per litter (12 * 3, n = 41, mean * SD) were similar to those observed the dams, the fetal enzyme activities did in litters from ad libitum-fed dams. Rapid not follow circadian rhythm patterns fetal growth during the time course of the (Figs. 2, 3). These enzyme activities did experiment was demonstrated by the in- increase during the 24-hr period. The increases in fetal body weight, total small creases appeared to be related closely to intestinal protein content, and small intes- the increases in the length of the small tinal length (Fig. 2). Fetal body weight intestine and somewhat less closely to inincreased linearly with time, B = 0.07, r2 creases in total intestinal protein content. = 83%. The total small intestinal protein Both sets of data versus time were subcontent, B = 0.10, and length, B = 0.11, jected to least-squares analysis for fitting increased somewhat more rapidly than did to a straight line. The resulting coeffithe body weight during this period. The cients of determination are shown in Table protein content increased in a linear man- 1. The enzyme activities, when related to ner, r2 = 82%. On the other hand, the length of the small intestine, were better elongation of the small intestine was not described by a straight-line function than well described by a linear function, r2 = when related to protein content, except for 47%. Less than 50% of the change in length lactase activity at pH 6, which was decan be attributed to a steady growth rate. scribed equally well by both relationships.
490
DEVELOPMENTAL
90r
1
BIOLOGY
VOLUME
60. 1977 DISCUSSION
feeding and dark
In the dams the activities of certain brush border enzymes, maltase, lactase, -100 - 92
-
6.4: 7.62 66~ 6.0: 524; - 4.4 2 - 3.6w 2.6 >20 f
7.0r 70. 6.2 5.4. 4.6 . 3.6. 3.0. %I 2.2 . 5
Mdl
5 4 3 2
I
1.2 ” -10: - 0.0 = - 0.6 5 - 0.4 ; -0.2 4 -0.0 z
.’
, , , , 1700 2100 0100 0500 0900 Time
, , 1300 1700
(EST)
2. Changes in leucylnaphthylamine hydrolyzing activity (LNAase), alkaline phosphatase (AP), small intestinal length (Sm. I. Ln.), small intestinal protein (Protein), and body weight (Body Wt.) in 19- to ZO-day-old fetuses. Mean 2 SE (n = 46). Least-squares determined line (- - -1. FIG.
The extremely low rz value of maltase activity at pH 3 was presumably due to the variability between litters at 0900 and 1300 hr. Coefficients of variation at these times were 42 and 44%, respectively. All of the fetal brush border enzyme activities appeared to increase at a considerably greater rate than did elongation of the small intestine (Figs. 2, 3). The brush border enzymes also increased in activity more rapidly than the intracellular lysosoma1 enzymes. In the graphs of the two disaccharidases, the activities at different pHs are graphed to the same scale in order to show the differences in slopes (Fig. 3). Considering that some of the enzyme activity at pH 6 is due to the presence of the lysosomal enzymes, the activity of the brush border enzymes would be increasing at an even greater rate than indicated.
Time
(EST1
3. Changes in y-glutamyltransferase (yGT), maltase (malt) activity measured at pH 6 and 3, and lactase (lact) activity measured at pH 6 and 3, in 19to 20-day-old fetuses. Mean + SE (n = 4-61. Leastsquares determined line (- - -). FIG.
TABLE VARIATION ACCOUNTED
1
IN FETAL ENZYME ACTIVITIES FOR BY THE FITTED REGRESSION
Coefficient of determination (r2)
Fetal enzyme
Leneth” Leucylnaphthylamine drolyzing activity Alkaline phosphatase y-Glutamyltransferase Lactase PH 6 PH 3 Maltase PH 6 DH 3
hy-
a Micromoles of substrate per length versus time. b Micromoles of substrate per protein versus time.
Proteinb
a7
69
79 72
64 32
79 77
78 61
76 63
56 17
hydrolyzed
per minute
hydrolyzed
per minute
491
BRIEF NOTES
leucylnaphthylamine hydrolyzing activity, and y-glutamyltransferase, follow circadian rhythms similar to those seen in male rats (Saito, 1972; Saito et al., 1976; Stevenson and Fierstein, 1976; Stevenson et al., 1975). The rhythm of alkaline phosphatase activity follows a slightly different periodicity. This is also seen in ad libitumfed male rats (Stevenson et al., 1975). In light of these similarities in enzyme activity patterns, it is assumed that, as in the male, these rhythms in the pregnant rat are cued chiefly or exclusively by the feeding schedule. In contrast, the activity patterns of these enzymes plus those of lysosomal maltase and lactase do not follow a circadian pattern in the 19- to 20-day-old fetuses. The possibility of endogenous rhythms of low amplitude and shortened cycle in these fetal enzymes is intriguing, but from this work it seems unlikely. During this stage of development the small intestine does not mature as a unified structure, but proliferation and differentiation of the villus surface occur at different times in different parts of the intestine (Kammeraad, 1942; Lindberg and Owman, 1966). It is reasonable to consider that the brush border enzymes of the absorptive cell would also be synthesized at different rates in the various areas of the intestine, and, thus, the nonlinear variation in enzyme activities of the whole intestine is a reflection of these differences. Presumably, the same processes cause the nonlinear variation in the lysosomal enzymes and in the elongation of the small intestine. Therefore, it may be fortuitous that the summation of the various processes in the epithelial cell surface and underlying cell layers results in a steady increase in total small intestinal protein content. By relating enzyme activity to intestinal length it was hoped to compensate for changes due to the general maturation processes and to leave only variability in
the enzyme activity. This additional variation is considered to result from random scatter and not from an endogenous rhythm. It is concluded that endogenous rhythms do not exist in the activity patterns of these brush border enzymes prior to birth. This observation coupled with the starvation and intravenous feeding studies done with adult rats leads to the further conclusion that intermittent and/or oral feeding not only cues these rhythms but is also involved in maintaining them. Since circadian rhythms are not present in fetal enzyme activities and do occur in the adult enzyme patterns, one may speculate about when these rhythms appear. If intermittent oral feeding is the only factor required to initiate these rhythms, then they would be expected to appear very soon after birth; by 6 to 6 days neonates suckle predominantly during the day (Walker et al., 1974). If other factors are involved, such as the changes in small intestinal structure and enzyme profile and the blood hormone levels which occur about the time of weaning (Deren, 1968), then circadian rhythmicity would not be apparent until after weaning. We thank Mrs. Lisa Wolff and Mr. Scot Zeigen for their technical assistance. This work was supported in part by Grant AM 18953 from the National Institute of Arthritis, Metabolic and Digestive ,Diseases and by a GRS-MSRP grant. REFERENCES DAHLQVIST, A. (1964). Method for assay of intestinal disaccharidases. Anal. Biochem. 7, 18-25. DEREN, J. J. (1968). Development of intestinal structure and function. In “Handbook of Physiology” (C. F. Code, ed.), Vol. 3, pp. 1099-1123. American Physiological Society, Washington, D.C. DOELL, R. G., and KRETCHMER, N. (1962). Studies on small intestine during development. I. Distribution and activity of P-galactosidase. Biochim. Biophys. Acta 62, 353-362. EICHHOLZ, A. (1967). Structural and functional organization of the brush border of intestinal epithelial cells. III. Enzymic activities and chemical composition of various fractions of Tris-disrupted
492
DEVELOPMENTAL BIOLOGY
brush borders. Biochim. Biophys. Actu 135, 475 482. FURUYA, S., and YUGARI, Y. (1971). Daily rhythmic change in the transport of histidine by everted sacs of rat small intestine. Biochim. Biophys. Actu 241, 245-248. FURUYA, S., and YUGARI, Y. (1974). Daily rhythmic change of L-histidine and glucose absorptions in rat small intestine in uiuo. Biochim. Biophys. Actu 343, 558-564. GALAND, G., and FORSTNER, G. G. (1974). Soluble neutral and acid maltases in the suckling-rat intestine. Biochem. J. 144, 281-292. GOLDBARG, J. A., and RUTENBERG, A. M. (1958). The calorimetric determination of leucine aminopeptidase in urine and serum of normal subjects and patients with cancer and other diseases. Cancer 1, 283-288. GOLZALEZ, A. W. A. (1932). The prenatal growth of the albino rat. And. Rec. 52, 117-138. KAMMERAAD, A. (1942). The development of the gastro-intestinal tract of the rat. J. Morphol. 70,323342. LINDBERG, T., and OWMAN, C. (1966). Intestinal dipeptidase. Development of dipeptidase activity in the small intestine of the rat as related to the development of the intestinal mucosa. Actu Physiol. Scam!. 68, 141-151. LLOYD, J. B., and WHELAN, W. J. (1969). An improved method for enzymic determination of glucose in the presence of maltose. Anal. Biochem. 30, 467-469. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurements
VOLUME 60, 1977
with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. NAFTALIN, L., SEXTON, M., WHITAKER, J. F., and TRACEY, D. (19691. A routine procedure for estimating serum y-glutamyltranspeptidase activity. Clin. Chim. Actu 26, 293-296. SAITO, M. (1972). Daily rhythmic changes in brush border enzymes of the small intestine and kidney in rat. Biochim. Biophys. Acta 286, 212-215. SAITO, M., MURAKAMI, E., and SUDA, M. (1976). Circadian rhythms in disaccharidases of rat small intestine and its relation to food intake. Biochim. Biophys. Actu 421, 177-179. SITREN, H., and STEVENSON, N. R. (1977). Rhythmic fluctuations in small intestinal functions, insulin, and gastrin in orally and intravenously fed rats. Gustroenterology 72, 1133. STEVENSON, N. R., and FIERSTEIN, J. S. (1976). Circadian rhythms of intestinal sucrase and glucose transport: cued by time of feeding. Amer. J. Physiol. 230, 731-735. STEVENSON, N. R., FERRIGNI, F., PARNICKY, K., DAY, S., and FIERSTEIN, J. S. (1975). Effect of changes in feeding schedule on the diurnal rhythms and daily activity levels of intestinal brush border enzymes and transport systems. Biochim. Biophys. Actu 406, 131-145. VILLEE, C. A., and HAGERMAN, D. D. (1958). Effect of oxygen deprivation on the metabolism of fetal and adult tissues. Amer. J. Physiol. 194, 457-464. WALKER, P. R., BONNEY, R. J., and POTTER, W. R. (1974). Diurnal rhythms of hepatic carbohydrate metabolism during development of the rat. Biothem. J. 140, 523-529.
