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Spermine and spermidine induce intestinal maturation in the rat
GASTROENTEROLOGY
1988;95:112-6
Spermine and Spermidine Induce Intestinal Maturation in the Rat C. DUFOUR,
G. DANDRIFOSSE,
N. ROMAIN,
and P. LEPOINT
Laboratory of Biochemistry, Department University of Lihge, Liege,. Belgium
P. FORGET, of Pediatrics,
In the present study, we aimed to induce precocious intestinal maturation in neonatal rats by the oral administration of polyamines. Groups of 5 rats received either saline, spermidine (10 pm01 daily), or spermine (6 pmol daily) orally on the lzth, 13th, and 14th postnatal days. The rats were killed on the 15th postnatal day. After the small bowel was removed, a l-cm distal ileal segment was removed for histologic examination and the remaining small bowel tissue was homogenized for further biochemical analysis. Polyamine administration was shown to induce structural and biochemical mucosal changes characteristic of postnatal maturation. Lactase, sucrase, and maltase specific activities (micromoles of substrate hydrolyzed per minute per gram of protein) were 80 f 10, 10 2 3, and 116 -I- 19 for the saline-treated rats; 51 f 7, 34 f 2, and 315 + 37 for the spermidine-treated rats; and 25 f 2, 46 + 5, and 419 f 63 for the spermine-treated rats, respectively. Similar results were obtained with rats, first treated with spermine (6 pmol) on the 7th postnatal day, receiving spermine (6 pmol) daily as described above and killed on the 10th postnatal day. Doseresponse experiments performed as reported above in rats whose treatment began on the 12th postnatal day showed that the maturational effects of orally administered spermine are dose-dependent.
I
ntestinal maturation in the rat is known to occur in the third postnatal week and is characterized by an increase in both epithelial cell proliferation and differentiation leading to histologic and biochemical changes in the small bowel epithelium (1,2). Before maturation, intestinal epithelial cells show large supranuclear vacuoles containing inclusion bodies. The latter disappear at the time of maturation. Biochemically, immature and mature enterocytes have different enzyme contents (3). Whereas some mucosal enzyme specific activities such as lactase decrease at the time of maturation, others such as
F. VERMESSE,
and Institute of Histology,
sucrase and maltase increase greatly during the same period (3). In a similar way, intestinal ornithine decarboxylase specific activity and polyamine concentrations increase at the time of maturation (4). The origin of these maturational changes is still a matter of intensive investigation. Polyamines (putrescine, spermidine, spermine) are ubiquitous low-molecular-weight polycationic compounds known to be involved in either cell proliferation or differentiation in many tissues (5). Although it has been suggested that polyamines are involved in the maturational process (4), experiments aimed at inducing precocious maturation using polyamines have not been reported. In the present study, we aimed to better evaluate the role played by polyamines in intestinal maturation by looking at the histologic and biochemical effects of orally administered polyamines on the small bowel mucosa of immature rats. Materials
and Methods
Experiments Initially Old Rats (Experiment
Performed 1)
on 12-Day-
On the 12th, 13th, and 14th postnatal days, three groups of 5 Wistar rats each received either saline (50 ~1, 9% NaCl), spermidine (5 pmol) in 50 ~1 of saline (9% NaCl), or spermine (3 pmol) in 50 ~1 of saline (9% NaCl) orally twice daily. During the treatment period, rats were returned to their mothers and normal feeding patterns were maintained. Animals were killed by decapitation on the 15th postnatal day. The small bowel was immediately removed, opened, and washed in cold saline. A l-cm piece of distal ileum was removed, fixed in formalin, and conventionally processed for histologic examination. The remaining small bowel was homogenized and aliquots were kept at - 70°C until analysis. Data from 36- and 60-day-old
0 1988 by the American
Gastroenterological 00X-5085/881$3.50
Association
I’OLYAMINES
untreated for adult
rats were obtained rats.
Experiments Old
Rats
and used
Initially
as reference
Performed
(Experiment
on 7-Day-
2)
of ornithine decarboxylase. Statistical
IVilcoxon’s nonparametric test was used to evaluate the significance of the differences beiween the mean results for different groups of rats.
Results
treated as received a spermine. day by in experi-
Experiments Old Rats
Experiment
iMethods
Protein content was determined according to the method of Bradford (6) using beef serum albumin as a standard. Deoxyribonucleic acid content was estimated according to the method described by Schneider (7) using calf thymrrs deoxyribonucleic acid (Boehringer-Mannheim, Mannheim, F.R.G.) as a standard. Disaccharidase activities were assayed by the method of Dahlqvist (8). Polyamine content was measured by high-performance
l‘nble
Initially
Performed
on 12-Day-
Detailed results of biochemical analyses performed in rats whose treatment began on the 12th postnatal day as described above (see the first section of Materials and Methods) together with adult rat reference values are shown in Table 1. Whereas protein content, deoxyribonucleic acid content putrescine content. and ornit hine decarboxylase specific activities were similar in all groups of rats, marked changes in hydrolasc specific activities and slight but significant changes in spermidine or spermine content occurred in spermidine- and spermine-treated groups when compared with the salinetreated control group. In these experiments, the results of which are reported in Table 1, we did not estimate the intestinal length of the rat pups. Other experiments (unpublished results), performed with la-day-old rats treated as described in the first section of Materials and Methods and killed on the 14th postnatal day, indicate that spermine could affect gut
Two groups of five lil-day-old rats were given either saline (50 ~1, 9% NaCI) or a single 6-pmol spermine dose orally (see Experiment 1) and killed on the 15th postnatal day. Intestinal tissue was processed as described above. Other
Analysis
Experiments
Six groups of five 12-day-old rats were described above (Experiment l), but each group different dose (0, 0.75, 1.5, 2.25, 3, or 4 pmol) of The animals were killed on the 15th postnatal decapitation. Further steps were as described ments 1 and 2. Single-Dose
113
liquid chromatography (9). Ornithine decarboxylase activity was assayed by the method of Russel and Snyder (10). The enzyme specific activities were expressed in micromoles of substrate hydrolyzed per minute per gram of protein in the case of disaccharidase and in picomoles of CO, produced per hour per milligram of protein in the case
values
On the i’th, 8th. and 9th postnatal days, two groups of 5 Wistar rats each received either saline (25 ~1) or spermine (3 pmol in 25 ~1 of saline) orally twice daily. The animals were killed by decapitation on the 10th postnatal day. Further steps were similar to those described for 1Z-day-old rats. Dose-Response
AN13 (;I r-1‘MAT1 IRATlON
I. Intestinal Biochemical Data for Control Rats and Rats First Treated Administered Spermidine or Spermine for 3 Days”
at 12 Days of Age With Orall!
Rat groups (II = 51
Biochemical
parameter
Protein content (mgig wet wt) DNA content (mg/g we:t wt) Sucrase specific activity hydrolyzedirnin activity
g protein)
(pmol maltose hydrolyzedimin Lactase specific activity
g protein)
(/.Lmol sucrose Maltase specific
(pm01 CO, produced/h
a Results
98.3 -c 2.4 3.1 -’ 0.2
10.3 -t 3.5
34.2 t 2.3”
80.2 1.4 10.6 9.0
t 19.3 i+ ? +
9.4 ?
mg protein] acid.
94.4 + 2.8 3.3 f 0.4
116.1
(pm01 lactose hvdrolyzedimin g protein) Putrescine content (pmolig protein) Spennidinc content (Fmolig protein) Spermine content (I*mol/g protein) Ornithine decarboxylase specific activity
DNA. deoxvribonucleic
Control (15 days old]
Spermidinetreated (15 days old)
are given
10.2 0.4 1.0 1.2 1.0
as mean
315.0 51.1 1.3 14.3 9.6
2 37.0” ? t + 2
7.4’ 0.3 0.2” 0.5
6.4 k 0.7
Sperminetreated (15 days old)
36 days
105.6 t 3.8 2.8 i 0.4
95.7 t- 1 7 3.5 i- 0 2
96.5 2 3.2
46.2
I? 5 1”
52.2 2 5.5”
400.3
+- 20.0”
46.4 -+ 5.1” 418.7 27.6 0.8 12.3 13.3
-+ 63.1” 2 2 -c -c
2.3” 0.1 0.3 0.3”
7.6 t 1.1
+ SEM. ’ p = 0.01 Wilcoxon’s
test).
Reference old
3.1 -+ 1.3” 1.3 t 0.1 13.3 i 0 3”
Values 60 days
320.3
+ 13.4”
3.6 2 0.2”
f3.9 r 0.3
9.8 -c 1.5
’ p = 0.020 (Ivilcoxon’s
old
test).
114
Table
DUFOUR
2.
ET AL.
GASTROENTEROLOGY
Intestinal Biochemical Data for Control Rats and Rats First Treated at 7 Days of Age With Orally Administered Spermine for 3 Days” Rat groups
Biochemical
Control (10 days old, saline-treated)
parameters
Protein content (mgig wet wt) DNA content (mgig wet wt) Sucrase specific activity (pmol sucrose hydroIyzedimin g protein) Maltase specific activity (km01 maltose hydrofyzedimin g protein) Lactase specific activity (FmoJ lactose hydrolyzed/ min g protein] Putrescine content (pmolig protein) Spermidine content (Fmolig protein) Spermine content (,bmol/g protein) DNA, deoxyribonucleic ’ p = 0.01 (Wilcoxon’s
acid. test).
89.2 k 4.6
(n = 5)
102.6 k 4.3 3.0 k 0.1
4.0 ? 1.4
40.6 k 6.@
t
14.1
decreased
sig-
Experiments
451.0 ? 36.2
80.5 L 8.6"
Experiment
After administration of a single spermine dose pmol) to l&day-old rats, a significant increase of sucrase (from 0.2 ? 0.2 to 21.5 ? 1.6 pmolimin g protein) and of maltase (from 70.7 t 4.2 to 217.3 t 10.9 kmolimin g protein] specific activities was observed in these rats on the 15th postnatal day. Lactase specific activities decreased significantly (from 67.9 t 4.2 to 43.6 rt 3.0 prnol lactose hydrolyzed per minute per gram of protein) in the same animals. (6
2.6 k 0.4
3.6 + 0.3
10.2 t 0.4
8.0 t 0.4"
10.2 k 0.6
14.3 + 1.2"
” Results
are given
as mean
2 SEM.
Changes
Structural mucosal growth: the value of protein content expressed in terms of gut length increased when compared with the value obtained in control rats. Sucrase and maltase specific activities (Table I] increased to adult levels in spermidineor sperminetreated rats, whereas lactase specific activities decreased significantly without reaching the characteristically low adult values. A significant increase of mucosal spermidine or spermine content compared with control rats was observed in rats treated with spermidine or spermine, respectively (Table 1).
Experiments Old Rats
of spermidine
As doses of orally administered spermine increased, a stepwise increase in maltase specific activities and a stepwise decrease in lactase specific activities were observed (Figure 1) for rats treated as described above (see third section of Materials and Methods). These effects seemed to level off at a daily dose of 6 pmol.
Single-Dose 175.3
that
Dose-Response
Sperminetreated (10 days old]
2.8 k 0.1
114.7 2 9.3
creased, whereas nificantly.
Vol. 95. No. 1
Initially
Performed
Induced
by Polyamines
Ileal villous enterocytes of neonatal salinetreated rats showed the presence of supranuclear vacuoles characteristic of immaturity [Figure 2A). A marked loss of these supranuclear vacuoles was observed in either spermineor spermidine-treated rats (Figure 2B). Disappearance of supranuclear vacuoles characteristically progressed from the base to the tips of the villi (Figure 2B). Apparent structural maturation occurred under the influence of orally administered polyamines.
on 7-Day-
Detailed results of biochemical analyses performed in rats whose treatment began initially on the 7th postnatal day as described above (see second section of Materials and Methods) are shown in Table 2. Conclusions drawn from the results obtained in these very young rats were similar to those obtained in 12-day-old rats. Whereas protein, deoxyribonucleic acid, and putrescine content did not change, sucrase and maltase specific activities increased to adult levels and lactase specific activities decreased significantly. The mucosal concentration of spermine in-
300
60-
LO-
too
ZO-
1.5
Figure
1. Effect
specific
3
L.5
6
8 ,urn
of spermine on intestinal maltase activities of spermine-treated rats.
spemxnofday
and
lactase
POLYAMINES
Figure
Discussion illthough intestinal maturation in neonatal rats has been the subject of intensive study by various authors, the factors triggering the major structural and biochemical changes occurring at the time of maturation are still poorly understood (ll13)
‘1‘0 obtain a better insight into the mechanisms involved. attempts have been made to induce precocious maturation by the exogenous administration of various intercellular messengers such as cortisone, insulin, and epidermal growth factor thyroxirll:. (12.131. ;ilthough the latter substances have been shown to induce precocious intestinal maturation to varving degrees. thev arc assumed to be modulators
ANI) (;111‘ MATIJKATIOn’
115
2. A. Distal ileum structure of control (saline-treated) rat. Enterocyte supranuclear vacuoles are present along the whole length of the villi. Periodic acid-Schiff reaction. H. Distal ileum structure of spermine-treated rat. Enteroc.ytes containing supranuclear vacuoles are localized to the tip of the villi. Mature enterocytes have replaced immaturt, cells elsewhere. Pericxlic: acid-Schiff reaction.
of an internal biological clock like thle key regulating factor (11). Besides hormonal factors, dietary constituents could also be involved in intestinal maturation (3). Our results show for the first time that, in the rat, orally administered polyamines induce precocious structural and biochemical intestinal maturation. The implications of our findings are that a changing dietary polyamine content could be, at least partly. responsible for the triggering of intestinal maturation. Additional data on the postnatal polyamine content of rat milk and pelleted rat diet are needed to answer this important question. Our results also show that mucosal spermine and spermidine levels are increased in spermineand spermidine-treated rats, respectively.
116
DUFOUR
ET AL.
Consequently, polyamine mucosal levels could possibly play a central role in the triggering of maturation. If this hypothesis is correct, the effects of maturation-inducing intercellular messengers could be mediated by polyamines. In this respect, both corticosterone (12) and epidermal growth factor (13) are known to increase ornithine decarboxylase activity, leading to increased polyamine synthesis and tissue differentiation. The maturation-inducing effects of the latter hormones could thus be due to increased mucosal polyamine levels. Although our results show that, like various hormones, orally administered polyamines have profound effects on intestinal maturation, they are probably modulating factors of an internal biological clock. As suggested in the literature, the rat intestinal cells might contain a “local timer” that switches on differentiation at the age of 21 days (11). Ornithine decarboxylase expression triggered by a genetically programmed modification of deoxyribonucleic acid transcription could, hypothetically, be the local timer that activates intestinal maturation. The latter enzyme is known to control polyamine synthesis and shows a sudden increase of activity on the 21st postnatal day in the rat small intestine (4). The ensuing mucosal polyamine concentration could be responsible for the events of differentiation that follow. The mechanisms whereby polyamines affect cellular proliferation and differentiation probably involve various effects at the level of cell deoxyribonucleic acid and ribonucleic acid. Alternatively, polyamines could act by increasing protein synthesis in an aspecific way. However, neither the decrease of lactase specific activity induced by polyamines nor the major structural changes observed would fit this latter hypothesis. Further studies are in progress to elucidate the mechanisms involved in the major maturation-inducing effects of polyamines.
GASTROENTEROLOGY
Vol. 95, No. 1
References 1. Klein
2.
3. 4.
5. 6.
10.
11. 12.
13.
RM, McKenzie JC. The role of cell renewal in the ontogeny of the intestine. I. Cell proliferation patterns in adult, fetal and neonatal intestine. J Pediatr Gastroenterol Nutr 1983;2:10-43. Klein RM, McKenzie JC. The role of cell renewal in the ontogeny of the intestine. II. Regulation of cell proliferation in adult, fetal and neonatal intestine. J Pediatr Gastroenterol Nutr 1983;2:204-28. Henning SJ. Role of milk-borne factors in weaning and intestinal development. Biol Neonate 1962;41:265-72. Luk GD, Marton LJ, Baylin SB. Ornithine decarboxylase is important in intestinal mucosal maturation and recovery from injury in rats. Science i980;2io:3ai-7. Janne J, Poso H, Raina A. Polyamines in rapid growth and cancer. Biochim Biophys Acta 1976;473:241-93. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54. Schneider WC. Determination of nucleic acids in tissues by pentose analysis. In: Colowick SP, Kaplan NO, eds. Methods in enzymology. Volume 3. New York: Academic, 1957:680-4. Dahlqvist A. Assay of intestinal disaccharidases. Anal Biothem 1968;22:99-107. Bontemps J, Laschet J, Dandrifosse G. Van Cutsem JL, Forget P. Analysis of dansyl derivatives of di- and polyamines in mouse brain, human serum and duodenal biopsy by highperformance liquid chromatography on a standard reversedphase column. J Chromatogr 1984;311:59-67. Russel DH, Snyder SH. Amine synthesis in rapidly growing tissues: ornithine decarboxylase activity in regenerating rat liver, chick embryo and various tumors. Proc Nat1 Acad Sci USA 1968;60:1420-7. Diamond JM. Hard-wired local triggering of intestinal enzyme expression. Nature 1986;324:408-9. Malo C, Menard D. Synergistic effect of insulin and thyroxin on the differentiation and proliferation of epithelial cells of suckling mouse small intestine. Biol Neonate 1953;44:177-84. Malo C, Menard D. Influence of epidermal growth factor on the development of suckling mouse intestinal mucosa. Gastroenterology i982;83:28-35.
Received September 36, 1987. Accepted February 22, 1988. Address requests for reprints to: Dr. P. Forget, Department of Pediatrics, CHU, Sart Tilman, 4999 Liege, Belgium. This work was supported by grant 3.4596.84 from the Fonds de la Recherche Scientifique Medicale.
1988;95:112-6
Spermine and Spermidine Induce Intestinal Maturation in the Rat C. DUFOUR,
G. DANDRIFOSSE,
N. ROMAIN,
and P. LEPOINT
Laboratory of Biochemistry, Department University of Lihge, Liege,. Belgium
P. FORGET, of Pediatrics,
In the present study, we aimed to induce precocious intestinal maturation in neonatal rats by the oral administration of polyamines. Groups of 5 rats received either saline, spermidine (10 pm01 daily), or spermine (6 pmol daily) orally on the lzth, 13th, and 14th postnatal days. The rats were killed on the 15th postnatal day. After the small bowel was removed, a l-cm distal ileal segment was removed for histologic examination and the remaining small bowel tissue was homogenized for further biochemical analysis. Polyamine administration was shown to induce structural and biochemical mucosal changes characteristic of postnatal maturation. Lactase, sucrase, and maltase specific activities (micromoles of substrate hydrolyzed per minute per gram of protein) were 80 f 10, 10 2 3, and 116 -I- 19 for the saline-treated rats; 51 f 7, 34 f 2, and 315 + 37 for the spermidine-treated rats; and 25 f 2, 46 + 5, and 419 f 63 for the spermine-treated rats, respectively. Similar results were obtained with rats, first treated with spermine (6 pmol) on the 7th postnatal day, receiving spermine (6 pmol) daily as described above and killed on the 10th postnatal day. Doseresponse experiments performed as reported above in rats whose treatment began on the 12th postnatal day showed that the maturational effects of orally administered spermine are dose-dependent.
I
ntestinal maturation in the rat is known to occur in the third postnatal week and is characterized by an increase in both epithelial cell proliferation and differentiation leading to histologic and biochemical changes in the small bowel epithelium (1,2). Before maturation, intestinal epithelial cells show large supranuclear vacuoles containing inclusion bodies. The latter disappear at the time of maturation. Biochemically, immature and mature enterocytes have different enzyme contents (3). Whereas some mucosal enzyme specific activities such as lactase decrease at the time of maturation, others such as
F. VERMESSE,
and Institute of Histology,
sucrase and maltase increase greatly during the same period (3). In a similar way, intestinal ornithine decarboxylase specific activity and polyamine concentrations increase at the time of maturation (4). The origin of these maturational changes is still a matter of intensive investigation. Polyamines (putrescine, spermidine, spermine) are ubiquitous low-molecular-weight polycationic compounds known to be involved in either cell proliferation or differentiation in many tissues (5). Although it has been suggested that polyamines are involved in the maturational process (4), experiments aimed at inducing precocious maturation using polyamines have not been reported. In the present study, we aimed to better evaluate the role played by polyamines in intestinal maturation by looking at the histologic and biochemical effects of orally administered polyamines on the small bowel mucosa of immature rats. Materials
and Methods
Experiments Initially Old Rats (Experiment
Performed 1)
on 12-Day-
On the 12th, 13th, and 14th postnatal days, three groups of 5 Wistar rats each received either saline (50 ~1, 9% NaCl), spermidine (5 pmol) in 50 ~1 of saline (9% NaCl), or spermine (3 pmol) in 50 ~1 of saline (9% NaCl) orally twice daily. During the treatment period, rats were returned to their mothers and normal feeding patterns were maintained. Animals were killed by decapitation on the 15th postnatal day. The small bowel was immediately removed, opened, and washed in cold saline. A l-cm piece of distal ileum was removed, fixed in formalin, and conventionally processed for histologic examination. The remaining small bowel was homogenized and aliquots were kept at - 70°C until analysis. Data from 36- and 60-day-old
0 1988 by the American
Gastroenterological 00X-5085/881$3.50
Association
I’OLYAMINES
untreated for adult
rats were obtained rats.
Experiments Old
Rats
and used
Initially
as reference
Performed
(Experiment
on 7-Day-
2)
of ornithine decarboxylase. Statistical
IVilcoxon’s nonparametric test was used to evaluate the significance of the differences beiween the mean results for different groups of rats.
Results
treated as received a spermine. day by in experi-
Experiments Old Rats
Experiment
iMethods
Protein content was determined according to the method of Bradford (6) using beef serum albumin as a standard. Deoxyribonucleic acid content was estimated according to the method described by Schneider (7) using calf thymrrs deoxyribonucleic acid (Boehringer-Mannheim, Mannheim, F.R.G.) as a standard. Disaccharidase activities were assayed by the method of Dahlqvist (8). Polyamine content was measured by high-performance
l‘nble
Initially
Performed
on 12-Day-
Detailed results of biochemical analyses performed in rats whose treatment began on the 12th postnatal day as described above (see the first section of Materials and Methods) together with adult rat reference values are shown in Table 1. Whereas protein content, deoxyribonucleic acid content putrescine content. and ornit hine decarboxylase specific activities were similar in all groups of rats, marked changes in hydrolasc specific activities and slight but significant changes in spermidine or spermine content occurred in spermidine- and spermine-treated groups when compared with the salinetreated control group. In these experiments, the results of which are reported in Table 1, we did not estimate the intestinal length of the rat pups. Other experiments (unpublished results), performed with la-day-old rats treated as described in the first section of Materials and Methods and killed on the 14th postnatal day, indicate that spermine could affect gut
Two groups of five lil-day-old rats were given either saline (50 ~1, 9% NaCI) or a single 6-pmol spermine dose orally (see Experiment 1) and killed on the 15th postnatal day. Intestinal tissue was processed as described above. Other
Analysis
Experiments
Six groups of five 12-day-old rats were described above (Experiment l), but each group different dose (0, 0.75, 1.5, 2.25, 3, or 4 pmol) of The animals were killed on the 15th postnatal decapitation. Further steps were as described ments 1 and 2. Single-Dose
113
liquid chromatography (9). Ornithine decarboxylase activity was assayed by the method of Russel and Snyder (10). The enzyme specific activities were expressed in micromoles of substrate hydrolyzed per minute per gram of protein in the case of disaccharidase and in picomoles of CO, produced per hour per milligram of protein in the case
values
On the i’th, 8th. and 9th postnatal days, two groups of 5 Wistar rats each received either saline (25 ~1) or spermine (3 pmol in 25 ~1 of saline) orally twice daily. The animals were killed by decapitation on the 10th postnatal day. Further steps were similar to those described for 1Z-day-old rats. Dose-Response
AN13 (;I r-1‘MAT1 IRATlON
I. Intestinal Biochemical Data for Control Rats and Rats First Treated Administered Spermidine or Spermine for 3 Days”
at 12 Days of Age With Orall!
Rat groups (II = 51
Biochemical
parameter
Protein content (mgig wet wt) DNA content (mg/g we:t wt) Sucrase specific activity hydrolyzedirnin activity
g protein)
(pmol maltose hydrolyzedimin Lactase specific activity
g protein)
(/.Lmol sucrose Maltase specific
(pm01 CO, produced/h
a Results
98.3 -c 2.4 3.1 -’ 0.2
10.3 -t 3.5
34.2 t 2.3”
80.2 1.4 10.6 9.0
t 19.3 i+ ? +
9.4 ?
mg protein] acid.
94.4 + 2.8 3.3 f 0.4
116.1
(pm01 lactose hvdrolyzedimin g protein) Putrescine content (pmolig protein) Spennidinc content (Fmolig protein) Spermine content (I*mol/g protein) Ornithine decarboxylase specific activity
DNA. deoxvribonucleic
Control (15 days old]
Spermidinetreated (15 days old)
are given
10.2 0.4 1.0 1.2 1.0
as mean
315.0 51.1 1.3 14.3 9.6
2 37.0” ? t + 2
7.4’ 0.3 0.2” 0.5
6.4 k 0.7
Sperminetreated (15 days old)
36 days
105.6 t 3.8 2.8 i 0.4
95.7 t- 1 7 3.5 i- 0 2
96.5 2 3.2
46.2
I? 5 1”
52.2 2 5.5”
400.3
+- 20.0”
46.4 -+ 5.1” 418.7 27.6 0.8 12.3 13.3
-+ 63.1” 2 2 -c -c
2.3” 0.1 0.3 0.3”
7.6 t 1.1
+ SEM. ’ p = 0.01 Wilcoxon’s
test).
Reference old
3.1 -+ 1.3” 1.3 t 0.1 13.3 i 0 3”
Values 60 days
320.3
+ 13.4”
3.6 2 0.2”
f3.9 r 0.3
9.8 -c 1.5
’ p = 0.020 (Ivilcoxon’s
old
test).
114
Table
DUFOUR
2.
ET AL.
GASTROENTEROLOGY
Intestinal Biochemical Data for Control Rats and Rats First Treated at 7 Days of Age With Orally Administered Spermine for 3 Days” Rat groups
Biochemical
Control (10 days old, saline-treated)
parameters
Protein content (mgig wet wt) DNA content (mgig wet wt) Sucrase specific activity (pmol sucrose hydroIyzedimin g protein) Maltase specific activity (km01 maltose hydrofyzedimin g protein) Lactase specific activity (FmoJ lactose hydrolyzed/ min g protein] Putrescine content (pmolig protein) Spermidine content (Fmolig protein) Spermine content (,bmol/g protein) DNA, deoxyribonucleic ’ p = 0.01 (Wilcoxon’s
acid. test).
89.2 k 4.6
(n = 5)
102.6 k 4.3 3.0 k 0.1
4.0 ? 1.4
40.6 k 6.@
t
14.1
decreased
sig-
Experiments
451.0 ? 36.2
80.5 L 8.6"
Experiment
After administration of a single spermine dose pmol) to l&day-old rats, a significant increase of sucrase (from 0.2 ? 0.2 to 21.5 ? 1.6 pmolimin g protein) and of maltase (from 70.7 t 4.2 to 217.3 t 10.9 kmolimin g protein] specific activities was observed in these rats on the 15th postnatal day. Lactase specific activities decreased significantly (from 67.9 t 4.2 to 43.6 rt 3.0 prnol lactose hydrolyzed per minute per gram of protein) in the same animals. (6
2.6 k 0.4
3.6 + 0.3
10.2 t 0.4
8.0 t 0.4"
10.2 k 0.6
14.3 + 1.2"
” Results
are given
as mean
2 SEM.
Changes
Structural mucosal growth: the value of protein content expressed in terms of gut length increased when compared with the value obtained in control rats. Sucrase and maltase specific activities (Table I] increased to adult levels in spermidineor sperminetreated rats, whereas lactase specific activities decreased significantly without reaching the characteristically low adult values. A significant increase of mucosal spermidine or spermine content compared with control rats was observed in rats treated with spermidine or spermine, respectively (Table 1).
Experiments Old Rats
of spermidine
As doses of orally administered spermine increased, a stepwise increase in maltase specific activities and a stepwise decrease in lactase specific activities were observed (Figure 1) for rats treated as described above (see third section of Materials and Methods). These effects seemed to level off at a daily dose of 6 pmol.
Single-Dose 175.3
that
Dose-Response
Sperminetreated (10 days old]
2.8 k 0.1
114.7 2 9.3
creased, whereas nificantly.
Vol. 95. No. 1
Initially
Performed
Induced
by Polyamines
Ileal villous enterocytes of neonatal salinetreated rats showed the presence of supranuclear vacuoles characteristic of immaturity [Figure 2A). A marked loss of these supranuclear vacuoles was observed in either spermineor spermidine-treated rats (Figure 2B). Disappearance of supranuclear vacuoles characteristically progressed from the base to the tips of the villi (Figure 2B). Apparent structural maturation occurred under the influence of orally administered polyamines.
on 7-Day-
Detailed results of biochemical analyses performed in rats whose treatment began initially on the 7th postnatal day as described above (see second section of Materials and Methods) are shown in Table 2. Conclusions drawn from the results obtained in these very young rats were similar to those obtained in 12-day-old rats. Whereas protein, deoxyribonucleic acid, and putrescine content did not change, sucrase and maltase specific activities increased to adult levels and lactase specific activities decreased significantly. The mucosal concentration of spermine in-
300
60-
LO-
too
ZO-
1.5
Figure
1. Effect
specific
3
L.5
6
8 ,urn
of spermine on intestinal maltase activities of spermine-treated rats.
spemxnofday
and
lactase
POLYAMINES
Figure
Discussion illthough intestinal maturation in neonatal rats has been the subject of intensive study by various authors, the factors triggering the major structural and biochemical changes occurring at the time of maturation are still poorly understood (ll13)
‘1‘0 obtain a better insight into the mechanisms involved. attempts have been made to induce precocious maturation by the exogenous administration of various intercellular messengers such as cortisone, insulin, and epidermal growth factor thyroxirll:. (12.131. ;ilthough the latter substances have been shown to induce precocious intestinal maturation to varving degrees. thev arc assumed to be modulators
ANI) (;111‘ MATIJKATIOn’
115
2. A. Distal ileum structure of control (saline-treated) rat. Enterocyte supranuclear vacuoles are present along the whole length of the villi. Periodic acid-Schiff reaction. H. Distal ileum structure of spermine-treated rat. Enteroc.ytes containing supranuclear vacuoles are localized to the tip of the villi. Mature enterocytes have replaced immaturt, cells elsewhere. Pericxlic: acid-Schiff reaction.
of an internal biological clock like thle key regulating factor (11). Besides hormonal factors, dietary constituents could also be involved in intestinal maturation (3). Our results show for the first time that, in the rat, orally administered polyamines induce precocious structural and biochemical intestinal maturation. The implications of our findings are that a changing dietary polyamine content could be, at least partly. responsible for the triggering of intestinal maturation. Additional data on the postnatal polyamine content of rat milk and pelleted rat diet are needed to answer this important question. Our results also show that mucosal spermine and spermidine levels are increased in spermineand spermidine-treated rats, respectively.
116
DUFOUR
ET AL.
Consequently, polyamine mucosal levels could possibly play a central role in the triggering of maturation. If this hypothesis is correct, the effects of maturation-inducing intercellular messengers could be mediated by polyamines. In this respect, both corticosterone (12) and epidermal growth factor (13) are known to increase ornithine decarboxylase activity, leading to increased polyamine synthesis and tissue differentiation. The maturation-inducing effects of the latter hormones could thus be due to increased mucosal polyamine levels. Although our results show that, like various hormones, orally administered polyamines have profound effects on intestinal maturation, they are probably modulating factors of an internal biological clock. As suggested in the literature, the rat intestinal cells might contain a “local timer” that switches on differentiation at the age of 21 days (11). Ornithine decarboxylase expression triggered by a genetically programmed modification of deoxyribonucleic acid transcription could, hypothetically, be the local timer that activates intestinal maturation. The latter enzyme is known to control polyamine synthesis and shows a sudden increase of activity on the 21st postnatal day in the rat small intestine (4). The ensuing mucosal polyamine concentration could be responsible for the events of differentiation that follow. The mechanisms whereby polyamines affect cellular proliferation and differentiation probably involve various effects at the level of cell deoxyribonucleic acid and ribonucleic acid. Alternatively, polyamines could act by increasing protein synthesis in an aspecific way. However, neither the decrease of lactase specific activity induced by polyamines nor the major structural changes observed would fit this latter hypothesis. Further studies are in progress to elucidate the mechanisms involved in the major maturation-inducing effects of polyamines.
GASTROENTEROLOGY
Vol. 95, No. 1
References 1. Klein
2.
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5. 6.
10.
11. 12.
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RM, McKenzie JC. The role of cell renewal in the ontogeny of the intestine. I. Cell proliferation patterns in adult, fetal and neonatal intestine. J Pediatr Gastroenterol Nutr 1983;2:10-43. Klein RM, McKenzie JC. The role of cell renewal in the ontogeny of the intestine. II. Regulation of cell proliferation in adult, fetal and neonatal intestine. J Pediatr Gastroenterol Nutr 1983;2:204-28. Henning SJ. Role of milk-borne factors in weaning and intestinal development. Biol Neonate 1962;41:265-72. Luk GD, Marton LJ, Baylin SB. Ornithine decarboxylase is important in intestinal mucosal maturation and recovery from injury in rats. Science i980;2io:3ai-7. Janne J, Poso H, Raina A. Polyamines in rapid growth and cancer. Biochim Biophys Acta 1976;473:241-93. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54. Schneider WC. Determination of nucleic acids in tissues by pentose analysis. In: Colowick SP, Kaplan NO, eds. Methods in enzymology. Volume 3. New York: Academic, 1957:680-4. Dahlqvist A. Assay of intestinal disaccharidases. Anal Biothem 1968;22:99-107. Bontemps J, Laschet J, Dandrifosse G. Van Cutsem JL, Forget P. Analysis of dansyl derivatives of di- and polyamines in mouse brain, human serum and duodenal biopsy by highperformance liquid chromatography on a standard reversedphase column. J Chromatogr 1984;311:59-67. Russel DH, Snyder SH. Amine synthesis in rapidly growing tissues: ornithine decarboxylase activity in regenerating rat liver, chick embryo and various tumors. Proc Nat1 Acad Sci USA 1968;60:1420-7. Diamond JM. Hard-wired local triggering of intestinal enzyme expression. Nature 1986;324:408-9. Malo C, Menard D. Synergistic effect of insulin and thyroxin on the differentiation and proliferation of epithelial cells of suckling mouse small intestine. Biol Neonate 1953;44:177-84. Malo C, Menard D. Influence of epidermal growth factor on the development of suckling mouse intestinal mucosa. Gastroenterology i982;83:28-35.
Received September 36, 1987. Accepted February 22, 1988. Address requests for reprints to: Dr. P. Forget, Department of Pediatrics, CHU, Sart Tilman, 4999 Liege, Belgium. This work was supported by grant 3.4596.84 from the Fonds de la Recherche Scientifique Medicale.