Peroxisomes and peroxisomal enzymes along the crypt-villus axis of the rat intestine

Differentiation (1993) 54:99-108 Ontogmy. hmpluir ud DiWrentirtiom Therapy

Q Springer-Verlag 1993

Peroxisomes and peroxisomal enzymes along the crypt-villus axis of the rat intestine Sylvie CablC Michele Kedinger ', Michel Dauqa ' ' Laboratoire dc Biologie Cellulaire du Developpement. Universite de Nancy I, Faculte des Sciences, BP 239. F-54506 Vandoeuvre-lesNancy Cedex, France INSERM,Unite 61, Biologie Cellulaire el Physiopathologie Digestives. 3, Avenue Moliere. IF-67300 Strasbourg. France Accepted in revised form April 8, 1993

Abstract. The development of peroxisomes and expression of their enzymes were investigated in differentiating intestinal epithelial cells during their migration along the crypt-villus axis. Sequential cell populations harvested by a low-temperature method were identified by microscopy, determination of alkaline phosphatase and sucrase activities and incorporation of [3H]-thymidine into DNA. Ultrastructural cytochemistry after staining for catalase activity, revealed the presence of peroxisomes in undifferentiated stem cells located in the crypt region. Morphometry indicated that the number of these organelles increased as intestinal epithelial cells differentiate. Catalase activity was higher in the crypt cells than in the mature enterocytes harvested from villus tips. On the other hand, an increasing gradient of activity was observed from crypts to villus tips for peroxisomal oxidases, i.e. fatty acyl coA oxidase, D-amino acid oxidase and polyamine oxidase. These findings indicate that biogenesis of peroxisomes occurs during migration of intestinal epithelial cells along the crypt-villus axis and that peroxisomal oxidases contribute substantially to the biochemical maturation of enterocytes.

Introduction

Since the first descriptions of microbodies (peroxisomes) in mouse proximal kidney tubules [67] and in rat hepatocytes [69], the electron microscopic features and the enzyme content of these subcellular organelles have been reported in detail in a number of animal and plant species and different tissues [9, 31, 43, 461. Peroxisomes are bound by one unit membrane and their size and abundance vary considerably depending on the cell type in which they are encountered [60]. These cytoplasmic corpuscles are characterized biochemically by the presence of H,O,-generating oxidases and a H,O,-destroying catalase [25]. Correspondence to: S. Cable

The diverse me:abolic pathways in which these structures are involved have been mainly investigated in mammalian hepat ocytes. Hepatic peroxisomes are involved in the biosynthesis of ether phospholipids [33, 341 dolichol, cholesterol [2, 821, and bile acids [45, 621. Moreover, peroxisomes play a role in the 8-oxidation of (very) long-chain fatty acids [47. 76, 77, 851 and in that of prostaglandins [22, 23, 701. Peroxisomes are also involved in the catabolism of polyamines [8,37], pipecolic acid [86], purines [58, 711 and glyoxylate [40, 571. Until now the precise role that peroxisomes play in enterocytes is not ye1 well defined. Ontogeny of the mammalian and amphibian intestine has provided natural systems in which the biogenesis of peroxisomes and expression of catalase have been examined. In the developing intestine of the mouse fetus, the number of peroxisomes and the catalase activity appeared to increase with time [14, 631. During spontaneous and thyroxine-induced metamorphosis of amphibian larvae, increases in frequency, size and catalase activity of peroxisomes have been reported in the differentiating secondary intestinal epithelium which replaces the degenerating larval (:primary) tissue [20, 211. These data suggest that a correlation exists between enterocytic differentiation and biogenesis of peroxisomes. The mammalian adult intestinal epithelium provides a unique model of cells a': different stages of differentiation aligned in an orderly pattern along the crypt-lumen axis. The epithelial cells lining the small intestinal mucosa are renewed every 2-3 days in rodents [13, 321. This renewal occurs b j continuous division of a stem cell population located in intestinal crypts, migration of daughter cells along the villus, and finally extrusion of senescent cells into the intestinal lumen [15, 321. As epithelial cells migrate up the crypt-villus axis they undergo functional and morphological differentiation and give rise to the variety of terminally differentiated phenotypes seen on the villus. This includes the development of welldefined apical microvillar brush borders and the maturation of a number of apical (e.g. alkaline phosphatase and sucrase) or basolateral (e.g. C a Z + and N a + / K + -

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ATPases) enzymes [56. 65, 83, 841. The participation of peroxisomes and of their enzymes in the structural and biochemical phenotype of the differentiating intestinal epithelial cells has not yet been investigated. The aims of the present study were two-fold: (a) to investigate the biogenesis of intestinal peroxisomes and (b) to assess the distribution of their enzymes along the cryptto-lumen regions of rat duodenal epithelium by the use of cytochemical and biochemical techniques. Methods Animals. Female and male Wistar rats weighing 200-250 g and 400-500 g, respectively, were housed in wire-floored cages and fasted 48 h before experiments with free access to water. Materials. Potassium cyanide (KCN) and phenol were purchased from Merck (Darmstadt. Germany). Flavin adenine dinucleotide (FAD) was obtained from Boehringer (Mannheim, Germany) and Caminoantipyrine was from Prolabo (Paris, France). Titanium oxysulfate was obtained from Touzart-Matignon (Paris, France). [3H]-thymidine was purchased from CEA (Saclay, France) and econofluor from NEN Research Products (Boston, Mass., USA). All other chemicals were obtained from Sigma Chemical (St. Louis, Mo., USA). Intestinal epithelial cell isolation. The low-temperature method described by Flint et al. [27] was modified to isolate sequentially epithelial cells along the crypt-villus axes of the rat intestine. Rats were killed with an overdose of chloroform. The small intestine was removed and rinsed thoroughly with an icecold 0.9% NaCl solution. A 15-cm long proximal segment was taken starting 2 cm from the pylorus, and then everted over a glass rod. The latter was removed and one end of the everted intestinal sac was ligated allowing to fill the sac with 10 ml of phosphate buffered saline (PBS), pH 7.4, containing 0.12 M NaCI, 1.2 m M MgSO,. 5 mM KCI, 1.2 mM KH2P04 and 10 mM Na,HPO, (buffer A). The other end of the everted intestinal segment was then ligated and the distended sac was immersed in 20 ml of buffer B containing 0.15 M NaCl and 2.5 mM EDTA, pH 7.4. Incubation was performed at 4O C under agitation in a rotating recipient. The buffer B was collected after time intervals of 10 min. Usually, 14 fractions were obtained. An aliquot of each cell fraction was controlled under a phase microscope. The remainder was centrifuged at 900g for 10 min at 4" C. Pelleted cells were washed twice with buffer A and used immediately for cytochemical studies or kept frozen at -80" C until enzyme assays.

In vivo incorporation of [3H]-thymidine into DNA. Rats were given an intraperitoncal injection of ['HI-thymidine (75 pCi per 100 g body weight; specific activity: 48 Ci/mmol) and killed 1 h later. intestinal epithelial cells were isolated and collected as described above, diluted in 5 vol. ultrapure water and homogenized at 4" C. Aliquots were used to measure DNA content according to the original spectrophotometric method of Burton [ l l ] modified by Giles and Myers [30]. Calf thymus DNA was used as standard. The incorporation of [3H]-thymidine into DNA was determined by counting 50 pl of the DNA containing filtrate in a pre-mixed liquid scintillation system (cconofluor). DNA synthesis was expressed as counts per minute (cpm) of 'H per microgram of DNA

WI. B m h border enzyme assays. Sucrase (EC 3.2.1.48) was measured according to the method of Dahlqvist [18] modified by Lloyd and Whelan [48] and alkaline phosphatase (EC 3.1.3.1) according to Eichholz (261. One unit (U) of enzyme activity corresponds to the amount of enzyme that hydrolyzes 1 pmol of substrate per minute; results were expressed as milliunits (mu) per milligram

protein. Proteins were assayed according to Lowry et al. [49] using bovine serum albumin as standard. Peroxisome enzyme assou. Cell fractions were homogenized in icecold bidistilled water with a glass-Potter homogenizer at 4" C. Homogenates were centrifuged at 700g for 10 min at 4" C. Triton X-100 was added to the supernatant (lY00;v/v) in order to release the peroxisomal enzymes [ 141. Catalase (EC 1.11.1.6) was measured according to the method of Baudhuin et al. [6]. Briefly, the assays were performed at 4" C for 0, 3 and 6 min in the reaction mixture (1060 pl) containing 20 mM imidazole-HCI buffer, pH 7, 0.1 'YObovine serum albumin, 1.66 m M hydrogen peroxide and the homogenate. The reaction was stopped by the addition of 600 pl of a 0.125% (w/v) saturated solution of titanium oxysulfate in 2 N H,SO, and the unhydrolyzed hydrogen peroxide was determined colorimetrically in this mixture by the extinction at 400 nm of the yellow peroxytitanium sulfate. The initial hydrogen peroxide concentration was measured similarly in the reaction mixture in which the homogenate was omitted. The breakdown of hydrogen peroxide follows a first order kinetic. Specific activity of catalase was expressed as 'milli-Units Baudhuin' (mUB) per mg of protein. D-amino acid oxidase (EC 1.4.3.3) and fatty acyl-coA oxidase (EC 1.3.99.3) were assayed by measurement of the H 2 0 2 production according to the method of Allain et al. [l], with slight modifications concerning the substrate concentrations. The reaction mixture contained in a total volume of 1 ml the following constituents: 50 mM potassium phosphate, pH 8,0.082 mM 4-aminoantipyrine, 1.06 mM phenol, 0.01 mM FAD, 0.8 iU of horseradish peroxidase and the homogenate. The reaction was carried out at 37" C by adding the appropriate substrate at the following optimal concentration: 50 mM D-proline for D-amino acid oxidase and 0.1 mM Iauroyl-coA (C: 12) or palmitoyl-coA (C: 16) for fatty acylcoA oxidase. The formation of H,O, was measured by following the increase in absorbance at 500 nm uersus a reagent blank. A molar extinction coeficient of 48.10' ml mol-' cm-' was used. A second assay for the acylcoA oxidase of the peroxisomal 8-oxidation system was performed using the modification reported by Hryb and Hogg [39] of the methods described by Cooper and Beevers [16] and Lazarow and de Duve [47]. The method was based on the increase in absorbance at 340 nm due to the reduction of nicotinamide adenine dinucleotide (NAD '). Assays were carried out at 37" C in the presence of KCN to prevent reoxidation of the NADH formed. The reaction mixture contained 50 mM phosphate bufTer at pH 7.4, 200 pM NAD', 100 pA4 Coenzyme A, 1 mM KCN, 12 m M dithiothreitol, 200 pg/ml bovine serum albumin, 0.025% Triton X-100 and 0.1 mM of the substrate (lauroylcoA or palmitoyl-coA). The reaction was started by adding the homogenate. A molar extinction coefficient of 63.9.105 ml mol-' cm-' was used. Spermine and spermidine oxidases, two polyamine oxidases (EC 1.5.3.3) were measured according to the method of Hayashi et al. [35] using N,-acetyl spermine (2.5 mM) and N,-acetyl spermidine (2.5 mM) as substrates, respectively. Specific activities of the peroxisomal oxidases were expressed as milliunits (mu) per mg of protein. The non parametric Wilcoxon test was used to compare peroxisome enzymes activities found in each cell population. Ultrastructural cytochernistry. Pelleted cells as well as rat duodenal segments were fixed in icecold 2% glutaraldehyde buffered with 0.1 M potassium phosphate, pH 7.4, for 1 h and washed in the same buffer for at least 1 h. In order to demonstrate the peroxidatic activity of catalase in pcroxisomes, samples were incubated for 2 h at 37" C in the alkaline 3,3' diaminobenzidine (DAB)-medium according to Novikoff et al. [59]. The DAB-medium was replaced after 1 h of incubation. Other samples serving as negative controls were treated with 0.2 M 3-amino-1, 2,4 triazole to inhibit catalase activity. After the incubation, samples were rinsed, post-fixed in 1% osmium tetroxide buffered with 0.1 M potassium phosphate,

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(1.3 cpm/pg DNA.) was significantly lower compared to

pH 7.4, for 30 min, dehydrated in ethanol and embedded in Araldite/epon mixture (v/Y). Semi-thin sections (1-2 pm) were prepared and stained with toluidine blue. Thin sections (SO-lo0 nm) were stained with uranyl acetate and lead citrate [66] and examined with a Philips CM12 electron microscope at 80 kV. Some thin sections were only stained with uranyl acetate. The omission of lead citrate staining permitted a better demonstration of catalase. activity in peroxisomes but resulted in a loss of contrast of the cellular structures. Six animals were studied for the morphological observations. The peroxisomal profiles were calculated per unit of area of enterocyte according to the methods outlined by Weibel [87]. Counting of peroxisomal profiles for statistical evaluation was performed directly in the electron microscope. The area of the corresponding cell was determined using micrographs taken at an initial magnification of 3000 and enlarged 2.3 times at printing. In vivo and in situ morphometry was performed on at least 30 cells for the determination of peroxisomal frequency and on a minimum of 100 DAB-positive structures for the measurement of their diameter. The mean number and mean diameter of pcroxisomes were statistically compared between the fractions according to the two-sided Wilcoxon signed rank test.

thymidine incorporation values measured in proliferative cell fractions harvested from lower crypt regions (21.6 cpm/pg DNA). Ultrastructural investigations showed that the five first fractions were predominantly composed of sheets of cells that remained attached by their apical tight junctions. These sheets of cells from the upper villi contained mature absorptive enterocytes with well-developed microvilli and a prominent terminal web (Fig. 2A) and contained also some goblet cells. Electron microscopic examination of their basal region revealed that the basement membrane was absent. Fractions 6-10 contained a higher proportion of isolated cells, showing intermediate phenotypes of differentiation. Indeed, microvilli were less developed at the apices of enterocytes (Fig. 2B). Cells of fractions 11-14 (Fig. 2C) were characterized by a rounded shape, a high nucleo-plasmatic ratio and by the abundance of free ribosomes. They mostly contained mitochondria which were distributed at random. Thus from these results, it appeared that the lowtemperature method with the distended intestinal sac yielded successfully villus tip cells and proliferating crypt cells and preserved their biochemical and structural cellular integrity. It may be emphasized that the mechanical shearing of whole 'denuded' villi, especially in the crypt fractions which frequently occurs when intestinal cell isolation is carried out at 37" C was not observed at

Results Isolation of intestinal epithelial cells

Fourteen cell fractions were harvested sequentially from the villus tips to the crypts of rat intestinal epithelium. Biochemical and morphological parameters were assessed in each cell fraction. Alkaline phosphatase and sucrase activities, expressed predominantly in villus tip cells [88], have been chosen as markers of intestinal cell differentiation. Therefore, alkaline phosphatase and sucrase activities were measured in total cellular lysates of each cell fraction. Alkaline phosphatase activity was tenfold higher in the first cell fractions than in the last ones (Fig. 1). A fivefold decrease was found for sucrase activity from villus tip cells to crypt cells (Fig. 1). Incorporation of [3H]-thymidine into intestinal epithelial populations showed a significant increase from villus to crypt cells (Fig. 1). Thus, incorporation of the labelled nucleoside into DNA of villus cell preparations VILLUS

4"

c.

On the basis of these data, cell fractions harvested along the intestinal crypt-villus axis were pooled into three categories. Group I was composed of fractions 1-5 enriched with mature cells of the upper villus. Fractions 6-10 were pooled in group 11. They included cells from the villus base and the upper crypt region. The last cell fractions 11-14 were pooled to yield group I11 enriched with middle and lower crypt cells. Visualization of peroxisomes

The cytochemical localization of the peroxidatic activity of catalase was analyzed a t the ultrastructural level to

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Fig. 1. Enzyme activity gradients and incorporation of tritiated thymidine into DNA along the crypt-villus axis. Results are the mean values obtained from four animals: 14 epithelial cell fractions were harvested

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Fig. 2. Ultrastructural examination of intestinal epithelial cells harvested along the crypt-villus axis according to the low-temperature isolation method. A Mature cells of the upper villus. Note the well-developed brush border (BB). the numerous mitochondria ( M ) and the absence of the basement membrane of these absorptive epithelial cells. B Cells of the upper crypt region. Microvilli (MI")were sparse and less developed than those of the villus region. C Cellular fraction of the crypt base. These proliferative cells were characterized by a rounded shape and a high nucleocytoplasmatic ratio. They contained some mitochondria. N. nucleus; Bars, 2 pm

trace the presence of peroxisomes in the different cell fractions. In order to verify to what extent the isolated fractions indeed represent villus or crypt cells the distribution and morphology of peroxisomes were also studied in vivo along the crypt-villus axis of the rat intestine. In all specimens examined the reaction product was limited to DAB-positive structures morphologically identifiable as peroxisomes. Diffusion of the reaction product outside of the organelles was never observed. In the control specimens treated with 3-amino-l,2,4triazole no reaction product was formed. In cells of group I, numerous DAB-positive structures were easily detected (Fig. 3 E). The distribution and morphology of these organelles were very similar to that of peroxisomes in enterocytes observed in situ at the villus tip (Fig. 3A). In those mature absorptive epithelial

cells, peroxisomes were mainly found in the region apical of the nucleus. They were observed in two forms: rounded to oval profiles and elongated, tubular structures. The mean diameter of the rounded bodies was slightly higher in isolated group I cells (0.20+0.06 pm) than in upper villus cells (0.17f0.05 pm) examined in vivo. In thicker sections, elongated worm-like structures were common with 0.25 f0.16 pm lengthwise and 0.08 0.02 pm wide (Fig. 3F). The mean numbers of peroxisomes converted on a unit area basis (100 pm') were 18.49f4.36 and 15.52f4.7 for isolated group I cells and in vivo villus tip enterocytes, respectively. In cells of group I1 (Fig. 3G) the mean diameter of peroxisomes was 0.17fO.06 pm. It was very similar to that of DAB-positive structures (0.17 fO.05) visualized in intestinal epithelial cells located at the villus base

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Fig. 3. Electron micrographs of the rat intestinal epithelial cryptvillus axis, in vivo (A-D) and of the intestinal epithelial cells isolated according to the low-temperature method and stained for catalasc activity by alkaline diaminobenzidine (DAB) method (EI). A Entcrocytes of the villus tip region. Numerous peroxisomes were detected in the supra nuclear region. B Enterocytes of the villus base region. Note less numerous peroxisomes present in these cells. C-D Cell of the crypt region. Some DABpositive structures

were detected. D Detail of a peroxisome. E-F Well-differentiated cell of the fractions (1-5). E Rounded peroxisomes were numerous. F Higher magnification of a tubular peroxisome. G Cells of the fractions (6-10) which contained some peroxisomes. H-I Undifferentiated cell of the fractions (11-14). I Detail of a peroxisome. BB, brush border; M Y , microvilli; N, nucleus; P, peroxisome. Burs, 1 pm (A, B, C, E, G, H); 0.2 pm (D, F, I)

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(Fig. 3B). The mean peroxisome numbers in group I1 cells (15.06 f 5.2) and in villus base epithelial cells (12.25f3.9rwere lower than that in group I cells and villus tip cells, respectively. The phenotype of group I11 cells (Fig. 3H)was very similar to that of stem cells observed in situ in the crypts (Fig. 3C). Both cells contained some DAB-positive structures. The latter exhibited a staining heterogeneity with a distinct electron-lucid halo between the matrix and the limiting membrane (Fig. 3 D, I). Their mean diameters were identical (0.16f0.04 pm). A slight but significant increase (P=O.OOl) was noted in the peroxisoma1 size from group 111 to group I cells. On the other hand, the diameter of DAB-positive structures did not exhibit significant variation from crypts to villus tips

Fig. 4. Means and standard deviations of peroxisomal enzymes activities measured in the three groups of intestinal epithelial cells harvested along the crypt-villus axis. I, Differentiated cells of the fractions (1-5); II, Cells of the fractions ( 6 1 0 ) ; III, Cells isolated of the crypt-base, fractions (1 1-14); n, number of animals studied; Ci, cyanide insensitive

in intestinal epithelial cells examined in situ. The mean numbers of peroxisomes were 11.65f5.28 and 8.31+ 5.14 in group I11 cells and in crypts cells, respectively. Significant differences were noted between cells of group I and I11 (P<0.05)and between crypt and villus tip cells (P
Figure 4 shows the distribution patterns of peroxisomal enzyme specific activities along the villus-crypt axis. Specific activities of peroxisomal oxidases were always higher in the group I than in group 111. Fatty acyl-coA oxidase specific activity was 3- and 4.5-fold higher in group

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I than in group 111 when lauroyl-coA (P
In this study the presence of peroxisomes and the level of peroxisomal enzymes were examined along the cryptvillus axis of adult rat small intestine using subpopulations of isolated epithelial cells. The low-temperature method described by Flint et al. [27] was chosen as its major advantage is to prevent possible deleterious effects compromising cellular integrity. Our morphometrical. cytochemical and biochemical results demonstrate that development of peroxisomes can be correlated with functional differentiation of enterocytes. Our electron microscopic investigations provide evidence that peroxisomes are already present in proliferating cells of the intestinal crypt region before they begin to differentiate. This observation is in good agreement with the previous work of Roels et al. [68]. Using cytochemistry at the ultrastructural level, these authors have occasionally observed peroxisomes in dividing cells of the human duodenal crypts. These data are of particular interest as the assembly and biogenesis of peroxisomes constitute a major and intriguing research topic. It has been recently assumed that new peroxisomes form by division of preexisting peroxisomes or in some cell types by budding out from the peroxisomal reticulum [10, 461. According to this model, partition of pre-existing peroxisomes is followed by the import of specific molecules in the daughter organelles giving rise to an increase in size. The fact that peroxisomes are present in intestinal crypt cells prior to their differentiation favour the latter hypothesis. While migrating from crypt to villus compartments, intestinal epithelial cells differentiate. This cellular process is accompanied by an increase in number of peroxisomes. The increase in peroxisomal size observed between cells isolated along the crypt-villus axis may be due to an artifact as the diameter of DAB-posiLive organelles did not vary markedly when intestinal epithelial cells were examined in situ from bottom to

top of the villus. The importance of these organelles in the metabolism of the mature enterocyte is emphasized by recent morphometrical studies [68] showing that the peroxisomal surface density - that is, total area of peroxisomal membrane expressed per cellular volume is 1.4-2.3 fold higher in enterocytes than in hepatocytes. A similar parallelism between peroxisomal biogenesis and cell differentiation has also been described during development. Indeed during embryonic and post-embryonic development of the vertebrate intestine the frequency and size of peroxisomes increase considerably in epithelial cells with their advanced stage of differentiation. A striking elevation of the number of peroxisomes is noted in the fetal mouse intestine at 17 days in utero, the highest concentration of these organelles is attained at 18 days, after which time their number becomes stable until 4 weeks after birth [14, 631. During spontaneous or triiodothyronine-induced amphibian metamorphosis, the intestinal epithelium is a kaleidoscope of cell death, proliferation and differentiation. The larval (primary) epithelium degenerates completely and is extruded in the gut lumen meanwhile basal stem cells proliferate and differentiate giving rise to the folded secondary epithelium of the newly-metamorphosed juvenile [19, 381. In this model the dekelopment of the secondary intestinal epithelium is accompanied by increases in frequency and size of peroxisomes [21]. Thus both studies indicate that the formation and augmentation of these organelles strictly parallel the process of differentiation, i.e. with the ability of cells to acquire their specific functions. In order to get a better insight into the metabolic functions in which rat intestinal peroxisomes are involved, we have studied the expression of peroxisomal enzymes in epithelial cells isolated along the crypt-villus axis using a low-temperature epithelial isolation method. We found that petoxisomes of rat intestinal epithelial cells are able to oxidize long fatty acids and that the three first enzymes involved in the peroxisomal 8-oxidation system displayed an increasing activity gradient from crypts to villus tips. Our data confirm and extend those previously reported. Indeed, it has already been shown that mucosa of guinea pig [78], rat [81] small intestine and of human colon [12, 751 possess enzyme activities for /?-oxidation of long- and very-long-fatty acids and that enzymes of fat metabolism exhibit a gradient activity along the villus axis [28, 29, 41, 44, 611. In rodent intestine the 8-oxidation system is greatly enhanced by peroxisome proliferators such as clofibrate [42, 791 or by a diet containing high levels of very-longchain-fatty acids [81]. Our biochemical study also reports for the first time on the presence of D-amino acid oxidase in rat intestinal epithelial cells. The function of this peroxisomal enzyme remains somewhat puzzling as D-amino acids are not physiologically produced in the animal cells. However they have been found in blood most probably originating from the intestinal flora [36]. D-amino acid oxidase is also suspected to contribute to gluconeogenesis in catalyzing the exergonic formation of a-keto acids. The latter are substrates for gluconeogenesis [25]. Thus, the increasing bottom- Lo-top gradient in D-amino acid oxi-

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dase that we observed from crypts to villus tips could reflect a progressive participation of rat intestinal epithelial cells to gluconeogenesis. Furthermore, our data revealed that activities of N,acetyl spermidine and spermine oxidases were detected in all subpopulations of epithelial cells harvested along the intestinal villus axis. Polyamine oxidases are peroxisoma1 enzymes as attested by biochemical [37] and cytochemical [8] studies. Their activities were always higher in the more mature villus cells and exhibited an increasing gradient from crypts to villus tips. A comparable gradient has also been reported for diamine oxidase [ 5 ] . The crypt-villus gradient of diarnine and polyamine oxidase activities suggests continued synthesis and activity of these enzymes involved in polyamine catabolism during intestinal cell migration toward the villus tip. Polyamines (putrescine, spermidine, spermine) are known to play an important role in normal and adaptive mucosal growth and in neoplastic intestinal cell proliferation [24, 731. Increases in polyamine biosynthesis mediated by the enzymes ornithine decarboxylase and S-adenosyl methionine decarboxylase have been reported in association with mucosal proliferation in the maturing small intestine of neonatal rats [53], regenerating mucosa after cytotoxic injury [53], intestinal adaptation postjejunectomy [50, 511 or during hormonal treatment 172). and refeeding 117, 52, 55, 801. Although this suggests that polyamine biosynthesis may occur in the proliferative intestinal crypt compartment, an increasing villus-crypt gradient is only found for S-adenosylmethionine decarboxylase activity and for the concentration of spermidine and spermine. Paradoxically an opposite gradient is observed for ornithine decarboxylase activity and putrescine concentration [3-5, 7, 52, 64, 741. Our finding that polyamine oxidase activities increase steadily from crypts to villus tips suggests that these enzymes play an important role in small intestinal epithelial cell differentiation by reducing the intraclllular concentrations of spermine and spermidine. Finally, we found that catalase activity was higher in the crypt cells than in the mature enterocytes harvested from villus tips. Our biochemical data agree with previous cytochemical findings indicating that although the number of peroxisomes in intestinal epithelial cells varies only slightly, the staining due to catalase activity appears weaker in the villus tip cells than in the villus base and crypt cells [68]. This lower catalase content probably reflects a normal turnover not compensated for by continuing synthesis when the cells grow older and move up the villus. Roels et al. [68] proposed that catalase synthesis takes place in the crypts up to the midvillus. In summary, results reported herein bring some insight about the roles exerted by peroxisomes in rat intestinal epithelial cells. Using a low-temperature epithelial isolation method we have shown clearly that high activities of peroxisomal /?-oxidation enzymes, polyamine oxidase and D-aminoacid oxidase are associated with the mature nonproliferating villus tip intestinal mucosal cells. These data allow the conclusion that mature intestinal cells are very active in /I-oxidation of very-long-

chain-fatty acids, in catabolism of polyamines and probably in gluconeogenesis. Furthermore we have demonstrated that a relationship exists between biogenesis of peroxisomes and differentiation of intestinal epithelial cells as the number and enzyme activities of the organelles increase steadily along the crypt-villus axis. It may be assumed that peroxisomal oxidases are good markers of maturity of intestinal epithelial cells. Acknodrdgrnerirs. The authors thank A. Stdkel for her excellent technical assistance. This work was supported by grants from INSERM. CNRS (SDI-12.43). ARC (N"6255). the Ligue Nationale contre le Cancer and the Fondation de la Recherche Medicale.

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