Life Sciences, Vol. 56, No. 19, pp. 16251630, 1995
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0024.3205(95)00129-8
MATURATION STATUS OF SMALL INTESTINE EPITHELIUM IN RATS DEPRIVED
OF DIETARY NUCLEOTIDES
Maria A. Ortega, Angel Gil and Antonio Sanchez-Pozo’
Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of
Granada, 18071 Granada, Spain
(Received in final form February 23, 1995)
Summarv
We describe the changes of several brush-border enzymatic activities
in different subpopulations of epithelial cells, separated sequentially
from the villus tip-to-crypt axis of the small intestine, induced by
deprivation of dietary nucleotides for different periods of time in
adult rats. Deprivation of dietary nucleotides lead to a decrease in
the content and specific activity of alkaline phosphatase, leucineaminopeptidase, maltase, sucrase and lactase in the villus tip, but
had little effect on the crypt zone. The effect of the nucleotide
deprivation on the enzymatic activity progressively increased
towards the tip of the villus. Since these enzymes are maturation
markers of the intestinal cells, these results support the idea that
dietary nucleotides affect the maturation status of small-intestine
epithelium.
Key Words: dietary nucleotides, intestinal brush-border enzymes, isolated enterocytes
The adaptive response of the small intestine to changes in food intake and dietary composition
may influence
the general digestive and absorptive function. The small intestine is extremely
food components. Most studies have focused on the effect
of overall protein, carbohydrate and fat, or, more specifically, on certain amino acids, sugars, or
fatty acids (1,2). More recently, we and others have reported that dietary nucleotides affect the
proliferation and maturation of the intestinal mucosa in young rats (3-6). Furthermore, nucleotides
may influence gene expression in the intestinal epithelium (7,8). Before conclusions can be drawn
about the effects of nucleotides on the maturation status of the intestinal cells, it is important to
measure maturation markers in pure preparations of these cells in order to exclude neighbouring
cells such as serosal and interstitial cells. Therefore, the aim of the present study was to
investigate whether the deprivation of dietary nucleotides produces changes in the enzymatic
markers of maturation in subpopulations of epithelial cells isolated sequentially from the villus tipto-crypt axis of rat jejunum.
sensitive to the presence of intraluminal
Methods
Exoerimental model.
Adult Wistar rats weighing 200 to 250 g were fed with either a semipurified synthetic diet
containing nucleotides (control group) or without nucleotides (deprived group). The composition
’ Corresponding author: A. SBnchez-Pozo. Department of Biochemistry and Molecular
Biology, Faculty of Pharmacy, University of Granada. 18071 Granada, Spain
1624
Nucleotides and Intestine Maturation
Vol. 56, No. 19, 1995
of the basic diet is shown in Table I. The nucleotide diet was obtained by supplementing 50 mg
of each of the following nucleoside monophosphates to 100 g of basic diet: AMP, GMP, IMP,
CMP and UMP. Nucleoside monophosphates were chosen on the basis of their high solubility.
The mixture gives a nucleotide content and composition similar to that of commercial chow, when
analyzed by HPLC (10).
TABLE I
Composition of the Basic Diet
Ingredients
Casein
Cornstarch
sugar
Cellulose
Soy oil
Mineral and Vitamin mix’
DL-Methionine
Choline bitartrate
Nucleotides
Amount (g/kg of diet)
167
506
150
50
100
23
3
1.1
N.D.*
’ From AIN Ad Hoc Committee on Standards for Nutritional Studies
(9). The mineral and vitamin mixture contained (g/kg of mix):
calcium diphosphate, 9.01; sodium chloride, 2.59; potassium citrate,
0.75; potassium phosphate, 5.61; potassium sulfate, 1.82;
magnesium sulfate, 3.02; manganous sulfate, 0.18; ferric lactate,
0.19; zinc carbonate, 0.056; cupric acetate x 2 H,O, 0.017;
potassium iodate, 3.5 x lOA; sodium selenite x 5 H20, 3.5 x 10d;
chromic potassium sulfate x 10 H,O, 0.019; calcium citrate, 0.71;
thiamine, 6 x 10 -3, riboflavin, 6 x 10m3;pyridoxine HCI, 7 x 10”;
nicotinic acid, 0.030; calcium panthotenate, 0.016; folic acid, 2 x
10m3;biotin, 2 x lOA; cyanocobalamin, 1 x lo-‘; retinol acetate, 1.4
x 10e3; cholecaiciferol, 2.5 x 10e5; DL-a-tocopherol, 5 x IO”;
phylloquinone, 5 x 10”.
“N.D. = not detectable when analyzed by HPLC.
Rats were weighed and killed on days 7, 14 and 21 of the dietary treatment. Exactly 20 cm
of the small intestine was taken from the Treitz angle (the length of the segment was measured
under a constant weight of 5 g), rinsed with ice-cold saline solution, and everted for cell isolation.
Cell isolation
The method, based on Weiser (1 l), used citrate to isolate epithelial cells from serosal and
interstitial cells. No proteases or other enzymes were used to dissociate cells. In brief, the
segments were incubated at 37°C for ten periods of 10 min each with an oxygenated solution of
phosphate-buffered saline (KH,P04 8 mM, NqHPO, 5.6 mM, KCI 1.5 mM, NaCl 96 mM, pH
7.3) containing sodium citrate 8 mM, 1.5 mM EDTA and 0.5 mM dithiothreitol. Agitation was
maintained at 80 cycles/ min in a water bath incubator. At the end of the different incubation
periods, the cells were collected by centrifugation at 500 rpm. Cell suspensions were maintained
at 37°C with oxygenation in plastic tubes to which CaCI, was added to make a final concentration
of 5 mM. This counteracted the chelating effects of EDTA and prevented disintegration of the
Vol. 56, No. 19, 1995
Nucleotides and Intestine Maturation
1625
brush border and loss of enzymes from the cell surface. The cells were centrifuged again, and the
pellet homogenized in 50 mM Tris-CIH, pH 7.4 and stored at -20°C until analysis.
Analvtical
Alkaline phosphatase was determined according to the method of Bergmeyer (12). Leucine
aminopeptidase was determined by the method of Maroux (13). Sucrase, maltase and lactase
activities were measured by the method of Dalhqvist (14). Enzymatic reactions were stopped in
the linear phase of color development. Total protein was determined by the method of Lowry (15)
using bovine albumin as a standard. DNA was determined fluorometrically by the method of
Labarca & Paigen (16) using calf thymus DNA as a standard. To determine the proliferation rate,
cells were treated by the detergent-trypsin method for the preparation of nuclei for flow cytometric
DNA analysis (17) and analyzed in a Becton-Dickinson flow cytometer. The results are expressed
as the percentage of dividing cells (mitotic index). The appearance of the everted intestine was
assessed by light microscopy in paraffin-embedded sections stained with hematoxylin and eosin
(18). Nucleotides were analyzed by reverse-phase HPLC following the method of Chomczynski
& Sacchi( 10).
Statistical analysis
Differences between means were tested for statistical significance by a two-ways analysis of
the variance (ANOVA) and Student-Newman-Keuls post hoc tests, using the PC-90 statistical
software package (BMDP Statistical Software, UCLA, USA). The program included time,
treatment, and time x treatment interaction. The probability level at which the differences were
considered significant was set at P< 0.05.
Results
The everted segments of the small intestine were studied by light microscopy at beginning and
at the end of the isolation process to ensure that there was no morphological alterations, and that
all the enterocytes had been removed without disrupting the underlying tissues (Figure 1). We did
not find significant differences in the morphology of the small intestine between the groups at any
time of treatment. Only some crypt bases and the luminapropriu persisted after the removal of
villus enterocytes from the medium, indicating that most of all enterocytes were obtained.
The cell gradient from villus tip-to-crypt axis defined by Weiser (11) for different enzymes and
proliferation rate was obtained. Mitotic index instead of thymidine kinase activity was used to
identify dividing cells. The results for each isolated fraction were presented as percentage of
isolated cells, calculated on the basis of the DNA content, to eliminate the variation in the villus
height between animals. These results, illustrated in Figure 2, validate our isolation procedure.
We have considered two different zones: the fractions where enzymatic activities were high
and proliferation rate low (O-65% isolated cells) designated as “villus tip” and the fractions where
enzymatic activities were low and proliferation rate high (65-100% isolated cells) designated as
“crypt zone”. As shown in Figure 2, there was a zone where both types overlapped each other.
This division of the intestine is consistent with the anatomical estructure of the intestine (19), and
has been used by several authors (11,20,21).
Vol. 56, No. 19,1995
Nucleotides and Intestine Maturation
1626
A
Fig. 1
Adult rat small intestine mucosa. Hematoxylin and eosin staining. A, before
starting cell isolation; B, after removal of the epithelial cells as described in
Methods.
Villus*
4
60
l
Crypt
/+ \
zone
\
i
\
4
20
40
60
80
100
% of Isolated
Cells
Fig. 2
Gradients of enzymatic activity and proliferation rate in intestinal isolated cells
from villus tip-to-crypt axis as described in Methods. The 100 % of cells
corresponds to the sum of the fractions expressed as DNA. Enzymatic activity is
expressed as U/mg DNA: (A) alkaline phosphatase, (x) leucine aminopeptidase,
( l) maltase, (m) sucrase, and (X) lactase. Proliferation rate is expressed as % of
dividing cells (*).
Figure 3 shows the effects of the dietary deprivation of nucleotides on alkaline phosphatase,
leucine aminopeptidase and maltase specific activities in different regions along the villus for
Vol. 56, No. 19, 1995
Nucleotides and Intestine Maturation
1621
different periods of time. The three enzymes were decreased in the nucleotide-deprived group,
especially leucine aminopeptidase. The decrease in specific activities affected all zones of the
villus tip without significant changes in the crypt zone. Minor changes were observed for sucrase
and lactase activities.
AP
I
LAP
60[ {-. \
MALTASE
.-\
% of Isolated Cells
Fig. 3
Enzymatic gradients for alkaline phosphatase (AP), leucine aminopeptidase (LAP)
and maltase from intestinal epithelial cells isolated from the villus tip-to-crypt axis
in control ( ---- ) and nucleotide-deprived (-)
rats at different times. Each
profile represents the mean of 4 analyses.
Nucleotides and Intestine Maturation
1628
Vol. 56, No. 19, 1995
Table II shows the effects of the nucleotide deprivation on total enzymatic content and specific
activity in the villus tip as a function of time. In general, all enzymatic activities were significantly
lower in the groups deprived of nucleotides than in the controls ones (F values for specific activity
and total enzyme content were respectively: 19.25 and 34.94, alkaline phosphatase; 79.62 and
63.94, aminopeptidase; 10.65 and 13.35, maltase; 13.59 and 5.65, sucrase; and 56.08 and 41.19,
lactase). Except for sucrase, there was a significant time-related decrease (F values expressed as
above were: 6.58 and 26.79, alkaline phosphatase; 36.77 and 10.98, aminopeptidase; 13.93 and
3.70, maltase, 0.10 and 1.97, sucrase; and 23.14 and 7.66, lactase). The df values were 1 for the
first source of variation (diet), 2 for the second (time) and 18 for total variation.
TABLE II
Effect of Dietary Nucleotide Restriction on Intestinal Villus Tip Enzymatic Activities
One week
Control
Deprived
Two weeks
Three weeks
Control
Deprived
Control
Deprived
Specific activities (mu/me Protein)
AP
173+_14
159f 17
153+31
65f17*f
117*10t
84f2”t
LAP
51+9
2851”
61+1
26fl*
61+3
25+1*
Maltase
106+ 16
995 12
80,27
57f9*t
97+14
61+18*f
Sucrase
27+4
20+2*
26k2
2121’”
29+ 1
18fl*
Lactase
4+1
2*1*
4+1
3$_1*
2+1t
2+1
Enzvme content (mU/mg DNA)
AP
492+49
321*97*
453f43
318+34”
481 -t58
243+ 17”s
LAP
257+28
111+75*
208f78
149f 10*
212+20
146+30*
Maltase
582+ 15
521+22*
5OOf75
364*94*t
572k12
369+80*t
Sucrase
181&54
98+7
209f46
114*44*
102+46
103+24
Lactase
24+5
13$-2*
26*4
13-t2*
14+1t,g
13+3
AP=Alkaline Phosphatase; LAP=Leucine Aminopeptidase.
Data are presented as the mean + S.D. (n=4).
Significant differences (P < 0.05): *, vs. Control; t, vs. one week: 0, vs. two weeks.
Our results indicate that dietary nucleotides affect the maturation status of the enterocytes. This
conclusion is based on the fact that dietary-nucleotide restriction induced a time-related decrease
in the content and specific activity of well known intestinal maturation markers in isolated
enterocytes. The effect of nucleotide deprivation was almost similar on enzymes present mainly
in the tip such alkaline phosphatase as on enzymes distributed along the villus such as Ieucine
aminopeptidase, maltase and sucrase (22), suggesting that nucleotides affect the entire maturation
process. The data presented in this paper confirm previous studies of intestinal mucosa in young
rats, which demonstrated that dietary nucleotides enhance intestinal growth (3) and intestinal repair
Vol. 56, No. 19, 1995
Nucleotides and Intestine Maturation
1629
after diarrhea or radiation (4-6). Our results expand these previous observations and suggest that
the effect of dietary nucleotides is important not only in young rats, where the effect has been
related to a high intestinal growth rate, but also in adult rats, where the growth rate is less. It
must be pointed out that the intestine presents a high cell turnover in adult rats (23). These results
also agree with studies performed with intestinal cell lines such as Caco-2 and IEC-6 cells that
have reported that nucleotides affect their proliferation and maturation (8).
The mechanism by which nucleotides influence maturation is not well understood. Dietary
nucleotides are known to play many biochemical and physiological roles, serving as precursors
for nucleic acid synthesis, being involved in protein synthesis and influencing lipid and lipoprotein
metabolism (24-26). Recently, we found that the levels of plasma apolipoprotein A-IV, which is
of intestinal origin, was increased by dietary nucleotide supplementation in newborn infants
(Pediatr. Res., in press). The present results could be interpreted as a result of an influence of
dietary nucleotides on the expression of brush-border enzymes, but further studies are needed.
Our results support the idea that dietary nucleotides are necessary nutrients for the intestine
(6,24). In contrast to other tissues such the liver, the intestine has a low capacity for the de ?rovo
synthesis of purines (27), and although the capacity for pyrimidine synthesis is high (28), this is
a slow and energy-demanding process. In addition, the nucleotide pools of the intestinal cells are
low compared to other tissues, suggesting that the intestine might suffer limitations of nucleotides
when these nucleotides are not present in the diet (29). Therefore, the use of dietary nucleotides
may be necessary to fulfil the nucleotide requirements. Indeed, dietary nucleotides are
incorporated by the intestine in considerable amounts (30). Nucleotide restriction might have
important repercussions in the digestive function as villi result immature. On the other hand,
excessive dietary nucleotide supplementation could have side effects such as the production of free
radicals of oxygen from the conversion of hypoxanthine to urate by xanthine oxidase (31).
Acknowledgments
We are grateful to E. Valverde, F. Ruiz-Cabello, M. Torres and D. Nesbitt for their valuable
help. This work was supported by grants from CICYT (Ministerio de Educacicin y Ciencia), and
Puleva, Spain.
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