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Am J Physiol Gastrointest Liver Physiol 293: G453-G460, 2007; doi:10.1152/ajpgi.00077.2006
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MUCOSAL BIOLOGY

Oral polyamine administration modifies the ontogeny of hexose transporter gene expression in the postnatal rat intestine

G. E. Wild,1 L. E. Searles,2 K. G. Koski,2 L. A. Drozdowski,3 J. Begum-Hasan,4 and A. B. R. Thomson3

1Division of Gastroenterology, Department of Medicine, and Department of Anatomy and Cell Biology, McGill University Health Center, and 2School of Dietetics and Human Nutrition, McGill University, Montreal, Quebec; 3Division of Gastroenterology, Department of Medicine, University of Alberta, Edmonton, Alberta; and 4Department of Paediatrics, Paediatric Endocrinolgy, Hotel Dieu Hospital and Queen's University, Kingston, Ontario, Canada

Submitted 21 February 2006 ; accepted in final form 11 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Gastrointestinal mucosal polyamines influence enterocyte proliferation and differentiation during small intestinal maturation in the rat. Studies in postnatal rats have shown that ornithine decarboxylase (ODC) protein and mRNA peak before the maximal expression of brush-border membrane (BBM) sucrase-isomaltase (SI) and the sugar transporters sodium-dependent glucose transporter 1 (SGLT1) and glucose transporter 2 (GLUT2). This study was undertaken to test the hypothesis that the oral administration of spermidine in postnatal rats upregulates the expression of ODC, thereby enhancing the expression of SI and SGLT1 in the brush-border membrane as well as basolateral membrane-facilitative GLUT2 and Na+-K+-ATPase. Northern and Western blot analyses were performed with antibodies and cDNA probes specific for SI, SGLT1, GLUT2, {alpha}1- and beta1-subunits of Na+-K+-ATPase, and ODC. Postnatal rats fed 6 µmol spermidine daily for 3 days from days 7 to 9 were killed either on postnatal day 10 (Sp10) or day 13 following a 3-day washout period (Sp13). Sp10 rats showed a precocious increase in the abundance of mRNAs for SI, SGLT1, and GLUT2 and Na+-K+-ATPase activity and {alpha}1- and beta1-isoform gene expression compared with controls. ODC activity and protein and mRNA abundance were also increased in Sp10 animals. The increased expression of these genes was not sustained in Sp13 rats, suggesting that these effects were transient. Thus, 3 days of oral polyamine administration induces the precocious maturation of glucose transporters in the postnatal rat small intestine, which may be mediated by alterations in ODC expression.1

ontogeny; sodium-dependent glucose transporter-1; glucose transporter-2; sucrase-isomaltase; orthinine decarboxylase

STUDIES using rodents have provided us with a framework for understanding the postnatal maturation of intestinal structure and function (18). During this dynamic period, changes in membrane properties and the expression and abundance of enzymes and nutrient transporters occur. A host of hormones, growth factors, and dietary factors may play a role, but the cellular mechanisms associated with these ontogenic changes have yet to be fully characterized.

Several studies have described the functional development of intestinal brush-border membrane (BBM) enzyme activity in the rat small intestine (29, 34, 43, 49). In rats, there is no BBM sucrase-isomaltase (SI) activity from birth until weaning (29). At weaning, a dramatic increase in SI activity occurs, with adult levels being rapidly established. Once weaning has occurred in rats, lactase-phlorizin hydrolase (LPH) activity declines, and this decline is associated with reduced LPH mRNA abundance (37).

There is less information available regarding the ontogeny of intestinal sugar transporters. Sodium-dependent glucose transporter 1 (SGLT1) is responsible for the uptake of glucose and galactose into the cell from the lumen (17), and Na+-K+-ATPase is responsible for maintaining the gradient necessary for its functioning (19, 42). Glucose exits the enterocytes via glucose transporter 2 (GLUT2), a sodium-independent facilitative transporter located on the basolateral membrane (BLM) (48). The signals and mechanisms involved in regulating intestinal gene expression are not completely understood. While there is evidence that glucocorticosteroids (38), thyroxine (32), and EGF (9) may play a role in regulating gene expression during the weaning period, diet does not appear to significantly affect this process (18).

Polyamines may be involved in the ontogeny of intestinal nutrient transport. Polyamines are present in rat colostrum and mature milk, suggesting that they could influence intestinal gene expression (41). There is a premature decline in LPH and a premature increase in SI following exogenous polyamine administration (1, 4, 12, 25, 26, 39, 53). The maturational changes in enzyme expression have been correlated to both mucosal polyamine levels and to the onset of the expression and activity of ornithine decarboxylase (ODC), the key enzyme in polyamine biosynthesis (31).

Accordingly, we proposed to test the hypothesis that the repeated oral administration of the polyamine spermidine in postnatal rats upregulates the expression and activity of ODC, thereby enhancing the abundance of SI, SGLT1, GLUT2, and Na+-K+-ATPase mRNA and protein levels. Thus, the purpose of the present work was to characterize the effects of exogenously administered spermidine on the time course of expression of these parameters in the postnatal rat small intestine. We choose to use a 6-µmol dose of spermidine based on the work of others (1, 4) as well as ourselves (54). This dose of spermidine is within the range of concentration detected for this polyamine in rat breast milk (42). The results suggest that the normal ontogenic expression of the digestion and absorption of glucose may be modified by an exogenous polyamine, spermidine, acting through the increase of ODC activity, which, in turn, enhances the mRNA and protein abundance of the enzymes and transporters involved in this process.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

Pregnant Sprague-Dawley rats (day 18 of gestation) were purchased from Charles River Canada (Saint Constant, QC, Canada). Principles for the care and use of laboratory animals approved by the Council in Animal Care were observed. This study received ethics approval from the Facility Animal Care Committee, Faculty of Medicine, of McGill University. The date of birth of the litter was designated as day 0, and the rat pups suckled until the time of the death. Dams were provided with water and standard Purina rat chow during the study period. All animals were maintained on a 12:12-h light-dark cycle (lights on 0700–1900 hours) in a climate-controlled environment (21°C).

To determine the effects of the polyamine spermidine, littermates were randomly assigned to receive either experimental oral feedings or placebo for 3 consecutive days starting on day 7 and ending on day 10. Suckling rats received either 8 µmol spermidine (Sigma Chemical) delivered in 25 µl of saline solution or an equivalent volume of saline (control) using a Gilson Pipetman (53). Animals did not receive spermidine or placebo during the 3-day washout period extending from postnatal days 10 to 12. Pups were killed on postnatal day 10 or after the washout period on day 13. The 3-day washout period was chosen to mirror the exposure time to spermidine to determine whether the effects of Sp were lasting or not. The washout period extended to day 13 (i.e., the time of death), which is far enough removed in time for the date of spontaneous weaning (i.e., days 18 to 20).

Rat pups from each group were killed by carbon dioxide-induced narcosis between 0800 and 1000 hours. Truncal blood was collected at the time of death, and the recovered serum was stored at –20°C until the time of assay. Forty centimeters of the proximal and distal small intestine beginning 2 cm adjacent to the ligament of Treitz and ileocecal valve, respectively, were flushed with ice-cold 0.9% saline. In postnatal rats, small intestinal segments were used. Small intestinal segments and mucosal scrapings were snap frozen in liquid nitrogen and stored at –70°C until the time of assay.

Preparation of Membrane Fractions and Enzyme Assays

Segments of the whole intestine from the proximal or distal small intestine (n = 4–6 in each group) were randomly pooled to obtain 1.0–1.2 g of tissue for the preparation of BBM or BLM fractions. Mucosal scrapings from the proximal and distal small intestine were pooled randomly for the preparation of membrane fractions. The full thickness of the neonatal intestine was used to obtain BBM and BLM fractions. We recognize that this is not ideal, as normally mucosal scrapings would be the starting material for this method. However, because of the young age of the animals, the fragility of their intestines, and the very small amounts of tissue available, we felt that it was the only way to proceed with this method. While one may speculate that the inclusion of the bowel wall in the preparation may interfere with the separation procedure or potentially contaminate the fractions, we carefully analyzed all samples for Na+-K+-ATPase and SI activity to ensure sample purity.

BLMs were isolated by a series of differential centrifugation steps, as described elsewhere (24). All steps were carried out at 4°C. Samples were homogenized using a Polytron homogenizer in a buffer containing 0.25 M sucrose, 150 mM NaCl, and 30 mM Tris (pH 7.5) in the presence of a protease inhibitor cocktail (5 µg/ml aprotinin, 2.5 µg/ml leupeptin, 0.5 µM/ml PMSF). The homogenate was centrifuged for 15 min at 6,000 g. The resulting pellet was rehomogenized in the same buffer and centrifuged for 15 min at 6,000 g. The supernatant was then centrifuged for 45 min at 45,000 g to yield a final crude BLM fraction, which was enriched ~20-fold for Na+-K+-ATPase activity and contained negligible SI activity. The final pellet was resuspended in homogenization buffer, and aliquots were stored at –70°C.

BBMs were isolated from the proximal and distal small intestine, as described elsewhere (27). All steps were performed at 4°C. Intestinal segments and mucosal scrapings were homogenized using a Polytron homogenizer (Brinkman Instruments) in an ice-cold buffer containing 500 mM mannitol and 10 mM HEPES buffer (pH 7.4) in the presence of the protease inhibitor cocktail described above. The homogenate was diluted sixfold with deionized water, to which 1 M MgCl2 was added to a final concentration of 10 mM. The suspension was agitated for 20 min and then centrifuged at 3,000 g for 15 min. The supernatant was centrifuged at 20,000 g for 30 min, and the resulting pellet was resuspended in a 100 mM mannitol and 10 mM HEPES (pH 7.4). The final pellet obtained after centrifugation at 20,000 g for 30 min was resuspended in 10 mM Tris (pH 7.4), and aliquots were stored at –70°C until the time of assay. Purified BBM fractions were enriched 12- to 16-fold for SI activity and contained negligible Na+-K+-ATPase activity.

SI was assayed according to the method of Dahlqvist (3). Na+-K+-ATPase activity was measured according to the method of Esmann (6).The reaction mixture contained 130 mM NaCl, 20 mM KCl, 4 mM MgCl2, 3 mM disodium ATP (Boehringer Mannheim), 30 mM histidine (pH 7.5), and 5 µg/ml membrane protein in a final volume of 1.0 ml. The reaction was carried out in the presence (i.e., residual ATPase) and in the absence (i.e., total ATPase) of 1 mM ouabain. Assays were performed in the presence of 1 mg sodium deoxycholate per milligram of protein. Protein was measured using the Bio-Rad reagent. Na+-K+-ATPase specific activity was expressed as nanomoles of inorganic phosphate liberated per minute per milligram of protein. Protein was measured using the Bio-Rad reagent. For ouabain inhibition curves, crude membranes were incubated for 2 h in the presence of varying concentrations of ouabain plus 1 mg sodium deoxycholate per milligram of protein. The Km of Na+-K+-ATPase for Na+ was determined by measuring Na+-K+-ATPase activity over a range of Na+ concentrations (0–200 mM), as previously described (54).

Quantitation of the number of Na+ pump molecules was by the method of "back door" phosphorylation of Resh (40), as described elsewhere (56). Crude membrane fractions (100 µg protein) were incubated for 30 min at room temperature in the presence of varying concentrations of H3PO4 (10–100 µM) and SDS (0.2 mg/mg protein) and with or without 2 mM ouabain in a final volume of 100 µl. The reaction was initiated by the addition of 10 µCi of carrier-free [32P]orthophosphate. The reaction was terminated by the addition of 1.0 ml of 20% trichloroacetic acid and 10% BSA in 100 mM H3PO4, and tubes were centrifuged for 2 min in a microfuge at 12,000 g. Pellets were washed three times in the same solution, solubilized in 300 µl of 0.1 N NaOH and 10% SDS, and then counted for radioactivity. After a correction for background counts, 70–85% of the signal was ouabain-stimulated binding of phosphate to Na+-K+-ATPase. The stoichiometry of the reaction is such that 1 mol Pi binds to 1 mol Na+-K+-ATPase. The concentration of Na+ pumps per milligram protein (i.e., pump abundance) was calculated and expressed as picomoles of Na+-K+-ATPase per milligram of protein. The pump turnover was expressed as nanomoles of Pi per minute.

ODC activity was assayed as previously described by Wild et al. (53). Aliquots of the final supernatant (250 µl) were incubated for 30 min at 37°C in the presence of 50 µM pyridoxal phosphate, 0.2 mM ornithine, and 0.5 µCi of L-[14C]ornithine (New England Nuclear, 51.3 µCi/µmol). The liberated 14CO2 was trapped on a piece of filter paper impregnated with 20 µl of 2 N NaOH, which was suspended in a center well above the incubation mixture. The reaction was terminated by the addition of trichloroacetic acid to a final concentration of 10%. Filter papers were transferred to 10 ml of scintillation cocktail, and the radioactivity was measured by scintillation spectroscopy. ODC activity is expressed as picomoles of 14CO2 released per hour per milligram of protein.

Immunoblot Analysis

Aliquots (100 µg protein) of BLMs for Na+-K+-ATPase {alpha}1- and beta1-subunits and GLUT2, of BBMs for SI and SGLT1, and of the final supernatant for ODC were separated by SDS-PAGE (7.5% gradient resolving gel) and transferred to nitrocellulose membranes. Alternatively, aliquots (15 µg protein) were immobilized on a nitrocellulose membrane using a slot-blot manifold (GIBCO-BRL) as previously described (54, 56). In each instance, the efficiency of protein transfer was verified by Ponceau S staining of the membranes. Nitrocellulose membranes were incubated overnight in Tris-buffered saline-0.5% Tween 20 (TTBS; pH 7.5) containing 5% (wt/vol) skim milk to block nonspecific protein binding sites. Membranes were then probed for 2 h at room temperature with rabbit anti-rat {alpha}1- or beta1-specific antisera (Upstate Biotechnology) to detect the 110-kDa {alpha}1 and a 55-kDa beta1 Na+-K+-ATPase isoform protein species (7, 30). The 120-kDa rat SI protein (21) was detected using a polyclonal antibody generously provided by Dr. Ward Olson (University of Wisconsin). The 74-kDa SGLT1 protein (17) was detected with an anti-SGLT-1 polyclonal antibody (raised against COOH-terminal amino acids 564–575 of the rabbit SGLT1 sequence, generously provided by Dr. K. Takata). Anti-GLUT2 (Biogenesis) polyclonal antiserum was used to detect a recognized 60-kDa GLUT-2 protein species (48). Anti-ODC antisera (generously provided by Dr. A. E. Pegg, Hershey, PA) was used to detect the 50-kDa protein species (22). Following incubation in primary antibody, membranes were washed briefly with TTBS (pH 7.5) to remove any unbound primary antibody. The reaction product was visualized after incubation for 1 h at room temperature with 125I-labeled goat anti-rabbit IgG (New England Nuclear, 1:1,500 dilution ) and X-ray exposure (Kodak XAR 5 film) for 24 h with an intensifying screen at –70°C. Molecular weights of the identified proteins were determined by a comparison with prestained molecular weight markers that were run on the same gels. Fluorograms were digitized with a Hewlett Packard Scanjet 4C and analyzed using Gel-Cypher Analyzer software (Lightools Research, Encinitas, CA), as described elsewhere (56). Signals were normalized to those present in adult controls fed chow, which were assigned a value of 1.0.

RNA Isolation and Northern and Slot-Blot Analyses

RNA was isolated from the small intestinal segments and mucosal scrapings according to the method of Chomczynski and Sacchi (2). Segments of the whole intestine from the proximal or distal small intestine (n = 4–6 in each group) were randomly pooled to obtain 1.0–1.2 g of either tissue for sample processing. Mucosal scrapings from the proximal and distal small intestine were randomly pooled from adult animals for the preparation of adult reference RNA fractions. Equal amounts (20 µg) of total RNA were denatured with formamide, fractionated on agarose-formaldehyde gels, and transferred to positively charged nylon membranes (Boehringer Mannheim) using conventional capillary blotting techniques. Ultraviolet inspection of the gel after ethidium bromide (10 µg/ml) staining confirmed both the presence of equivalent amounts of 18S and 28S rRNA per lane as well as the integrity of the RNA. The RNA for slot-blot analysis was treated in a similar manner. Samples containing 6 µg RNA were blotted on a nylon membrane (Boehringer Mannheim) using a slot-blot manifold (GIBCO BRL), as previously described (54, 56).

Northern and slot-blot analyses were performed using the following cDNA probes: the 3.7-kb {alpha}1-subunit and 2.7-kb beta1-subunit Na+-K+-ATPase transcripts (30) were detected with the corresponding 0.79-kb EcoR1-Pst1 {alpha}1 and 1.2-kb Nco-Stu beta1 fragments (kindly obtained from Dr. J. Lingrel, University of Cincinnati). The 6.0-kb SI transcript (21) was detected using a 0.85-kb EcoRI SI fragment (kindly obtained from Dr. Peter Traber of the University of Pennsylvania Medical Center). The 2.1-kb EcoRI SGLT1 fragment (kindly obtained from Dr. N. O. Davidson, University of Chicago) was used to detect the 4.5-kb SGLT1 transcript (17). A 2.4-kb EcoRI GLUT2 fragment (kindly obtained from the American Tissue Culture Collection, Rockville, MD) was used to detect the 2.8-kb GLUT2 transcript (48). A 1.9-kb EcoRI ODC fragment (kindly obtained from Dr. Perry J. Blackshear, Howard Hughes Institute) was used to detect the 2.6- and 2.2-kb transcripts (46).

cDNA probes were labeled with digoxigenin-labeled dUTP (Boehringer Mannheim) by the random primer procedure described by Feinberg and Vogelstein (8). The hybridization protocol has been described elsewhere in detail (55). Fluorograms were digitized with a Hewlett-Packard Scanjet 4C and analyzed using Gel-Cypher Analyzer software (Lightools Research, Encinitas, CA). Each blot was additionally probed with GAPDH cDNA (American Type Culture Collection), a constitutively expressed transcript, to normalize for minor variations in the amount of RNA loaded per sample. Thus, signal densities of the RNA blots were first normalized by dividing signal density of the experimental blots by the signal density of the GAPDH loading control. These normalized values were expressed as a relative percentage of the value determined in controls (designated 100%) (56). For slot-blot analysis, a minimum of four fluorograms was evaluated for each data point. Finally, RNA preparations of the proximal and distal small intestine from control and spermidine-treated rats were analyzed at the same time to control for intra-assay variability.

Serum Cortisol Analysis

Levels of serum cortisol were measured in serum samples from placebo- and spermidine-fed rats using the DSL-2000 radioimmunoassay kit (Diagnostic Systems Laboratories, Webster, TX) according to the manufacturer's instructions. Standards as well as unknown samples were assayed in triplicate. Scatchard plots were constructed from these data to obtain cortisol concentrations of each unknown sample, which were compared with the accepted normal range of values for postnatal day 10–13 rats.

Data Analysis

Results were expressed as means ± SE. The placebo day 10 arm was treated as baseline, and changes were compared with this. One-way ANOVA was used to determine the significance of the postnatal age effect, and two-way ANOVA was used to determine postnatal age and treatment effects. The statistical significance of the differences between groups was determined by ANOVA. Differences between group means were analyzed by Duncan's multiple-comparison procedure. The significance level was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of Spermidine

Enzyme and transporter expression. The effects of spermidine administered orally for 3 consecutive days (i.e., from days 7 to 9) on the abundance of SI, SGLT1, and GLUT2 mRNA and protein were examined in the proximal and distal small intestine (Fig. 1, A–C). Levels of SI, SGLT1, and GLUT2 mRNA and protein measured in day 10 spermidine-treated rats were higher (P < 0.05 in each case) than the corresponding levels measured on days 10 and 13 in placebo-treated rats and on day 13 in spermidine-treated animals. Thus, the effects of spermidine on SI and hexose transporter expression were not sustained in those rats that underwent a 3-day washout prior to death at day 13.


Figure 1
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  		Fig. 1. Effects of spermidine on the expression of sucrase-isomaltase (SI; A), sodium-dependent glucose transporter 1 (SGLT1; B), and glucose transporter 2 (GLUT2; C) mRNA abundance and protein in the proximal and distal small intestine on postnatal days 10 and 13. Slot blots were prepared from RNA, and membrane fractions were isolated from the proximal and distal small intestine and probed with SI, SGLT1, and GLUT2 cDNA and SI antibody, respectively. Signals were quantitated and normalized as described in MATERIALS AND METHODS, and normalized values are expressed as a relative percentage of the value determined in adult controls fed chow (designated as 100%). Each bar represents the mean ± SE from 4–6 determinations. Representative slot blots of SI, SGLT1, and GLUT2 mRNA and protein in the proximal and distal small intestine of placebo- and spermidine-treated rats are shown at the bottom. *Significant differences between day 10 spermidine-treated rats versus the other groups (P < 0.05).

 
Na+-K+-ATPase {alpha}1- and beta1-subunit mRNA and protein levels. The effects of spermidine on Na+-K+-ATPase activity and Na+ pump abundance in the proximal and distal small intestine were examined (Fig. 2A). The daily administration of spermidine for 3 consecutive days prior to death on day 10 elicited a precocious expression of Na+-K+-ATPase activity and Na+ pump abundance in both regions of the small intestine. Levels of Na+-K+-ATPase activity and Na+ pump abundance in the proximal and distal small intestine in day 10 spermidine-treated rats were greater (P < 0.05 in each case) than the corresponding levels of these parameters measured in day 10 and 13 placebo-treated rats as well as in day 13 spermidine-treated animals. Thus, the effects of spermidine on Na+-K+-ATPase activity and Na+ pump abundance were transient and were not sustained during the washout period. The expression of these parameters at day 13 did not differ significantly from placebo-treated neonatal rats killed at day 13. The pattern of alterations of Na+-K+-ATPase activity and Na+ pump abundance observed after spermidine treatment was paralleled by a corresponding precocious induction of Na+-K+-ATPase {alpha}1- and beta1-subunit mRNA and protein levels in both regions of the small intestine (Fig. 2, B and C). Again, the effects of spermidine on these parameters were transient.


Figure 2
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  		Fig. 2. Effects of spermidine on Na+-K+-ATPase activity and Na+ pump abundance (A) as well as Na+-K+-ATPase {alpha}1-subunit (B) and beta1-subunit (C) mRNA and protein in the proximal and distal small intestine. Slot blots were prepared from RNA, and membrane fractions were isolated from the proximal and distal small intestine and probed with {alpha}1- and beta1-subunit cDNA and {alpha}1- and beta1-subunit antibody, respectively. Signals were quantitated and normalized as described in MATERIALS AND METHODS, and normalized values are expressed as a relative percentage of the value determined in adult controls fed chow (designated 100%). Each bar represents the mean ± SE from 4–6 determinations. Representative slot blots of {alpha}1- and beta1-subunit mRNA and protein in the proximal and distal small intestine of placebo- and spermidine-treated rats are shown at the bottom. *Significant differences between day 10 spermidine-treated rats versus the other groups (P < 0.05).

 
ODC activity and mRNA and protein levels. The levels of ODC activity (Fig. 3A) as well as ODC mRNA and protein (Fig. 3B) were measured in the proximal and distal small intestine in postnatal rats fed spermidine for 3 consecutive days and killed on day 10. The levels of ODC activity and mRNA and protein abundance were significantly (P < 0.05 in each case) greater in day 10 spermidine-treated rats compared with the day 10 and 13 placebo-treated groups and the day 13 spermidine-treated group. Thus, the effects of spermidine were temporary, as the levels of ODC activity and mRNA and protein abundance measured in both regions of the small intestine at day 13 did not differ significantly from those measured in placebo-fed rats killed at this time point.


Figure 3
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  		Fig. 3. Effects of spermidine on the expression of orthinine decarboxylase (ODC) activity (A) and mRNA (B) and protein (C) abundance in the proximal and distal small intestine on postnatal days 10 and 13. Slot blots were prepared from RNA, and membrane fractions were isolated from the proximal and distal small intestine and probed with ODC cDNA and ODC antibody, respectively. Signals were quantitated and normalized as described in MATERIALS AND METHODS, and normalized values are expressed as a relative percentage of the value determined in adult controls fed chow (designated 100%). Each bar represents the mean ± SE from 4–6 determinations. Representative slot blots of ODC mRNA and protein in the proximal and distal small intestine of placebo- and spermidine-treated rats are shown at the bottom. *Significant differences between day 10 spermidine-treated rats versus the other groups (P < 0.05).

 
Finally, exposure of postnatal rats to spermidine had no effect on the yields of BLM and BBM protein, mucosal RNA, or serum cortisol concentrations (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Effects of Spermidine on Na+-K+-ATPase and Glucose Transporter Expression

The oral administration of spermidine for 3 days from days 7 to 10 resulted in a precocious expression of SI, SGLT1, and GLUT2 mRNA and protein in the proximal and distal small intestine on day 10 (Fig. 1, A–C). These findings extend the earlier observations pertaining to the induction of SI activity by spermidine (1, 4, 12, 25, 26, 39, 53). The findings reported here are the first description of the effects of polyamines on SI, Na+-K+-ATPase, and glucose transporter mRNA and protein levels. However, we did not measure sugar uptake in this study, as it is unknown if the changes in mRNA and protein reflect attractions in sugar transporter activities. The previously observed spermidine-induced increases in SI and Na+-K+-ATPase activities reflect corresponding alterations in mRNA and protein levels, which in turn define some of the cellular processes associated with this phenomenon.

The administration of spermidine for 3 days resulted in the enhancement of Na+-K+-ATPase activity, Na+ pump abundance, and {alpha}1- and beta1-subunit mRNA and protein levels in the proximal and distal small intestine on day 10 (Fig. 2, A–C). Our findings pertaining to the induction of SGLT1, GLUT2, and Na+-K+-ATPase expression suggest that the effects of spermidine are not restricted to disaccharidases but also affect the transporters that mediate glucose uptake and exit across the enterocyte BBM and BLM domains, respectively. While earlier studies have provided evidence to suggest that glucocorticoids modulate the developmental profile of Na+-K+-ATPase activity (20, 57), our findings provide the first evidence for the induction of Na+-K+-ATPase gene expression by polyamines.

It is unclear from the present findings whether spermidine has direct effects on SI, glucose transporter, and Na+-K+-ATPase gene expression or whether the effects are mediated by a spermidine-induced release of gut hormones or growth factors. Our earlier findings, together with the present work, failed to uncover any spermidine-induced changes in plasma cortisol levels (53). This is in contrast to the findings reported elsewhere of polyamine-mediated increases in the serum cortisol concentration (25, 26, 39). These observations are in keeping with an indirect mode of action of polyamines, which may involve the stimulation of ACTH release. While it is tempting to speculate that surges in endogenous glucocorticoids following exposure to exogenous polyamines may have a permissive effect on SI, glucose transporter, and Na+-K+-ATPase gene expression in the neonate, it is unlikely that spermidine-induced persistent elevations in plasma glucocorticoids are solely required to enhance gene expression.

The reversion to lower levels of SI, SGLT1, GLUT2, and Na+-K+- ATPase following the withdrawal of spermidine treatments between days 10 and 13 (Figs. 1 and 2) may reflect decreased mucosal growth and/or a dependence on exogenous polyamines for growth following 3 consecutive days of spermidine exposure. These findings suggest a possible adaptation to exogenous spermidine, whereby in the absence of an exogenous source of polyamines the mucosa of the suckling rat small intestine becomes unable to produce the levels of polyamines required to sustain normal intestinal mucosal growth and maturation.

Effects of Spermidine on ODC Expression

The effects of spermidine exposure on ODC activity and mRNA and protein abundance in the suckling rat small intestine have not been described previously. In view of the importance of ODC in enterocyte growth and differentiation (23, 33, 44) and the precocious SI, glucose transporter, and Na+-K+-ATPase expression observed here following spermidine exposure, we sought to examine the effects of in vivo administration of this polyamine on ODC gene expression. Exposure to spermidine over a 3-day period induced significant increases in ODC activity and gene expression in both the proximal and distal small intestine (Fig. 3, A–C). These findings extend our earlier observations of increased ODC activity following a 3-day exposure to spermidine starting on postnatal day 7 (53). Other studies, however, have reported disparate findings with regard to ODC activity following exposure of suckling rats to exogenous polyamines (1, 4). It is noteworthy that in these studies, where ODC is either unchanged or decreased, the 3-day exposure to polyamine was initiated much later (i.e., after day 12) in the postnatal period compared with days 7–9 of the present study. It is possible that the immature small intestine is more responsive to the precocious induction of ODC by polyamines when exposure is initiated prior to day 10, as was the case in the present study.

The expression of ODC is downregulated by exogenous polyamines in a variety of mature, fully differentiated cell types (14, 36, 45). Most of these data are derived from in vitro studies. In the present study, we observed in vivo increases in ODC activity and expression, which parallels the appearance of mature villous enterocyte that occurs under the influence of orally administered spermidine, as previously reported by our laboratory (54) and other investigators (1, 4). Mature villous enterocytes have been shown to have higher levels of ODC activity and expression relative to immature enterocytes (22).

The increased levels of ODC expression observed in the present study may reflect the increased numbers of mature enterocytes lining the small intestinal villi. Support for this contention comes from earlier findings of increased ODC activity in villous compared with crypt enterocytes, suggesting that ODC is a marker of the differentiated state (22).

The physiological ligand and signaling pathways that regulate ODC expression, and the early nuclear events triggered by ODC induction, await further definition. Putative ligands and signaling mediators include gastrin, EGF, short-chain fatty acids, and PKC as well as the early response genes c-myc, c-fos, and c-jun (14, 15, 51). The observation that the peak time for ODC expression is earlier than for SI, glucose transporter, and Na+-K+-ATPase (day 14 vs. day 21) suggests that ODC may be an important signal for developmental gene regulation. The expermiments with spermidine were not designed to measure the relative time courses for the onset of gene expression. However, based on these data, we speculate that early development and the administration of polyamines signal the upregulation of ODC, which then leads to the enhanced expression of genes involved in glucose transport.

A potential shortcoming of this study is that there are no morphological or functional data associated with this study. We recognize that alterations in gene expression or protein abundance do not always correlate with changes in functional parameters. However, because of the short treatment period used in this study, we were interested in the effects at the mRNA/protein level. We agree that measurements of morphological or functional changes in the intestine would add significantly to the impact of the study; however, we suggest that these parameters be the focus of future work using longer treatment periods.

Speculations

The developmental profiles of expression of the transporters that mediate glucose absorption during the postnatal period are similar and parallel the ontogenic profile of SI expression. These findings suggest that SI and enterocyte hexose transporters work in concert to facilitate the digestion and absorption of glucose. The earlier appearance of ODC activity and mRNA and protein raises the possibility that there may be an early response gene that is associated with the initiation of the absorptive phenotype. This argument is strengthened further by the observation that the polyamine-associated upregulation of the disaccharidase and transporter mRNA and protein remains coordinated and, once again, is associated with changes in ODC gene expression. These alterations occur by a mechanism that does not appear to involve variations in the serum cortisol concentration. This would underscore the need for the definition of the signaling pathways that might link changes in ODC expression to the maturation of absorptive function seen during postnatal development.


   GRANTS
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 MATERIALS AND METHODS
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This work was supported by operating grants from the Dairy Farmers of Canada and the Crohn's and Colitis Foundation of Canada. G. E. Wild is a senior research scholar of Les Fonds de la Recherche en Sante du Quebec.


   ACKNOWLEDGMENTS
 
The technical assistance of N. Sauriol and J. A. Thomson is gratefully appreciated. We thank G. Ingram for expert word processing skills.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. B. R. Thomson, Zeidler Ledcor Bldg., 130 Univ. Campus, Univ. of Alberta, Edmonton, AB, Canada T6G 2X8 (e-mail: alan.thomson{at}ualberta.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supplemental material for this article is available online at the American Journal of Physiology-Gastrointestinal and Liver Physiology website. Back


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  1. Buts JP, DeKeyser N, Kolanowski J, Sokal E, Van Hoof F. Maturation of villus and crypt cell functions in rat small intestine: role of dietary polyamines. Dig Dis Sci 38: 1091–1098, 1993.[CrossRef][ISI][Medline]
  2. Chomczynski P, Sacchi N. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987.[ISI][Medline]
  3. Dahlqvist A. Assay of intestinal disaccharidase. Anal Biochem 22: 99–107, 1968.[CrossRef][ISI][Medline]
  4. Dufour C, Dandrifosse G, Forget P, Vermesse F, Romain N, Lapoint P. Spermine and spermidine induce intestinal maturation in the rat. Gastroenterology 95: 112–116, 1988.[ISI][Medline]
  5. Duluc I, Jost B, Freund J. Multiple levels of control of the stage- and region-specific expression of rat intestinal lactase. J Cell Biol 123: 1577–1586, 1993.[Abstract/Free Full Text]
  6. Esmann M. ATPase and phosphatase activity of Na,K-ATPase: molar and specific activity determination. Methods Enzymol 156: 105–124, 1988.[ISI][Medline]
  7. Fambrough DM, Lemas MV, Hamrick M, Emerick M, Renaud KJ, Inman EM, Hwang B, Takeyasu K. Analysis of subunit assembly of the Na-K-ATPase. Am J Physiol Cell Physiol 266: C579–C589, 1994.[Abstract/Free Full Text]
  8. Feinberg AP, Vogelstein BA. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13, 1983.[CrossRef][ISI][Medline]
  9. Foltzer-Jourdainne C, Garaud JC, Nsi-Emvo E, Raul F. Epidermal growth factor and the maturation of intestinal sucrase in suckling rats. Am J Physiol Gastrointest Liver Physiol 265: G459–G466, 1993.[Abstract/Free Full Text]
  10. Freeman HJ, Quamme GA. Age-related changes in sodium-dependent glucose transport in rat small intestine. Am J Physiol Gastrointest Liver Physiol 251: G208–G217, 1986.[Abstract/Free Full Text]
  11. Freeman TC, Wood IS, Sirinathsinghji DJ, Beechey RB, Dyer J, Shirazi-Beechey SP. The expression of the Na+/glucose cotransporter (SGLT1) gene in lamb small intestine during postnatal development. Biochim Biophys Acta 1146: 203–212, 1993.[Medline]
  12. Georges P, Dandifosse G, Vermesse F, Forget P, Deloyer P, Romain N. Reversibility of spermine-induced intestinal maturation in the rat. Dig Dis Sci 35: 1528–1536, 1990.[CrossRef][ISI][Medline]
  13. Giannella RA, Orlowski J, Jump ML, Lingrel JB. Na+-K+-ATPase gene expression in rat intestine and Caco-2 cells: response to thyroid hormone. Am J Physiol Gastrointest Liver Physiol 265: G775–G782, 1993.[Abstract/Free Full Text]
  14. Ginty DD, Marlowe M, Pekala PH, Seidel ER. Multiple pathways for the regulation of ornithine decarboxylase in intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 258: G454–G460, 1990.[Abstract/Free Full Text]
  15. Groblewski GE, Ways DK, Seidel ER. Protein kinase C regulation of IEC-6 cell ornithine decarboxylase. Am J Physiol Gastrointest Liver Physiol 263: G742–G749, 1992.[Abstract/Free Full Text]
  16. Hecht A, Torbey CF, Korsmo HA, Olsen WA. Regulation of sucrase and lactase in developing rats: role of nuclear factors that bind to two gene regulatory elements. Gastroenterolgy 112: 803–812, 1997.[CrossRef][ISI][Medline]
  17. Hediger MA, Rhoads DB. Molecular physiology of sodium-glucose cotransporters. Physiol Rev 74: 993–1026, 1994.[Free Full Text]
  18. Henning SJ, Rubin DC, Shulman RJ. Ontogeny of the intestinal mucosa. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by Johnson LR. New York: Raven, 1994.
  19. Horisberger JD, Lemas V, Kraehenbuhl JP, Rossier BC. Structure-function relationship of Na, K-ATPase. Annu Rev Physiol 53: 565–584, 1991.[CrossRef][ISI][Medline]
  20. Horvath K, Blochin B, Hill I, Verma R, Lu RB, Lebenthal E. The pre- and postnatal development of Na+/K+-ATPase in gastrointestinal organs of the rat: effect of betamethasone treatment. J Pediatr Gastroenterol Nutr 16: 412–418, 1993.[ISI][Medline]
  21. Hunziker W, Speiss M, Semenza G, Lodish HF. The sucrase-isomaltase complex: primary structure, membrane orientation, evolution of a stalked, intrinsic brush border protein. Cell 46: 227–234, 1986.[CrossRef][ISI][Medline]
  22. Johnson LR, Tseng CC, Wang P, Tipnis UR, Haddox MK. Mucosal ornithine decarboxylase in the small intestine: localization and stimulation. Am J Physiol Gastrointest Liver Physiol 256: G624–G630, 1989.[Abstract/Free Full Text]
  23. Johnson LR, McCormack SA. Regulation of gastrointestinal mucosal growth. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by Johnson LR. New York: Raven, 1994.
  24. Jorgensen PL. Purification of Na,K-ATPase: enzyme sources, preparative problems, preparation from mammalian kidney. Methods Enzymol 156: 29–46, 1988.[ISI][Medline]
  25. Kaouass M, Deloyer P, Dandrifosse G. Intestinal development in suckling rats: direct or indirect spermine action? Digestion 55: 160–167, 1994.[CrossRef][ISI][Medline]
  26. Kaouass M, Sulon J, Deloyer P, Dandrifosse G. Spermine-induced precocious intestinal maturation in suckling rats: possible involvement of glucocorticoids. J Endocrinol 141: 279–283, 1994.[Abstract]
  27. Kessler M, Acuto O, Storelli C, Murer H, Muller MJ, Semenza G. A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Biochim Biophys Acta 506: 136–154, 1978.[Medline]
  28. Krasinski SD, Estrada G, Yeh KY, Yeh M, Traber PG, Rings EHHM, Buller HA, Verhave M, Montgomery RK, Grand RJ. Transcriptional regulation of intestinal hydrolase biosynthesis during postnatal development in rats. Am J Physiol Gastrointest Liver Physiol 267: G584–G594, 1994.[Abstract/Free Full Text]
  29. Leeper LL, Henning SJ. Development and tissue distribution of sucrase-isomaltase in rats. Am J Physiol Gastrointest Liver Physiol 258: G52–G58, 1990.[Abstract/Free Full Text]
  30. Lingrel JB, Orlowski J, Shull M, Price EM. Molecular genetics of Na, K-ATPase. Prog Nucleic Acid Res Mol Biol 38: 37–89, 1990.[ISI][Medline]
  31. Luk GD, Marton LJ, Baylin SB. Ornithine decarboxylase is important in intestinal mucosal maturation and recovery from injury in rats. Science 210: 195–198, 1980.[Abstract/Free Full Text]
  32. McDonald MC, Henning SJ. Synergistic effects of thyroxine and dexamethasone on enzyme ontogeny in rat small intestine. Pediatr Res 32: 306–311, 1992.[ISI][Medline]
  33. McCormack SA, Johnson LR. Role of polyamines in gastrointestinal mucosal growth. Am J Physiol Gastrointest Liver Physiol 260: G795–G806, 1991.[Abstract/Free Full Text]
  34. Martin GR, Henning SJ. Enzymic development of the small intestine: are glucocorticoids necessary? Am J Physiol Gastrointest Liver Physiol 246: G695–G699, 1984.[Abstract/Free Full Text]
  35. Marton LJ, Pegg AE. Polyamines as targets for therapeutic intervention. Annu Rev Pharmacol Toxicol 35: 55–91, 1995.[CrossRef][ISI][Medline]
  36. Miyamoto KI, Hase K, Taketani Y, Minami H, Oka T, Nakabou Y, Higihira H. Developmental changes in intestinal glucose transporter mRNA levels. Biochem Biophys Res Commun 183: 626–631, 1992.[CrossRef][ISI][Medline]
  37. Motohashi Y, Fukushima A, Kondo T, Sakuma K. Lactase decline in weaning rats is regulated at the transcriptional level and not caused by termination of milk ingestion. J Nutr 127: 1737–1743, 1997.[Abstract/Free Full Text]
  38. Nanthakumar N, Henning SJ. Ontogeny of sucrase-isomaltase gene expression in rat intestine: responsiveness to glucocorticoids. Am J Physiol Gastrointest Liver Physiol 264: G306–G311, 1993.[Abstract/Free Full Text]
  39. Nsi-Emvo E, Chaton B, Foltzer-Jourdainne C, Gosse F, Raul F. Premature expression of sucrase isomaltase triggered by corticoid-dependent changes in polyamine metabolism. Am J Physiol Gastrointest Liver Physiol 270: G54–G59, 1996.[Abstract/Free Full Text]
  40. Resh MD. Quantitation and characterization of the (Na+, K+)-adenosine triphosphatase in the rat adipocyte plasma membrane. J Biol Chem 257: 11946–11952, 1982.[Free Full Text]
  41. Romain N, Dandrifosse G, Jeusette F, Forget P. Polyamine concentration in rat milk and food, human milk, and infant formulas. Pediatr Res 32: 58–63, 1992.[ISI][Medline]
  42. Schultz SG, Hudson RL. Biology of sodium-absorbing epithelial cells: dawning of a new era. In: Handbook of Physiology. The Gastrointestinal System. Intestinal Absorption and Secretion. Bethesda, MD: Am. Physiol. Soc., 1991, sect. 6, vol. IV, chapt. 2, p. 45–81.
  43. Seetharam B, Yeh KY, Moog F, Alpers DH. Development of intestinal brush border membrane proteins in the rat. Biochim Biophys Acta 470: 424–436, 1977.[Medline]
  44. Seidel ER, Scemama JL. Gastrointestinal polyamines and regulation of mucosal growth and function. J Nutr Biochem 8: 104–111, 1997.[CrossRef][ISI]
  45. Shu R, David ES, Ferraris RP. Dietary fructose enhances intestinal fructose transport and GLUT5 expression in weaning rats. Am J Physiol Gastrointest Liver Physiol 272: G446–G453, 1997.[Abstract/Free Full Text]
  46. Tappenden KA, Thomson ABR, Wild GE, McBurney MI. Short chain fatty acids increase proglucagon and ornithine decarboxylase messenger RNAs following intestinal resection in rats. J Parenter Enteral Nutr 20: 357–362, 1996.[Abstract]
  47. Thomson ABR, Wild GE. Adaptation of intestinal nutrient transport in health and disease. Dig Dis Sci 42: 453–488, 1997.[CrossRef][ISI][Medline]
  48. Thorens B. Glucose transporters in the regulation of intestinal, renal and liver glucose fluxes. Am J Physiol Gastrointest Liver Physiol 270: G541–G553, 1996.[Abstract/Free Full Text]
  49. Vaeth GF, Henning SJ. Postnatal development of peptidase enzymes in rat small intestine. J Pediatr Gastroenterol Nutr 1: 111–117, 1982.[ISI][Medline]
  50. Walker WA, Sanderson IR. Epithelial barrier function to antigens. An overview. Ann NY Acad Sci 664: 10–17, 1992.[CrossRef][ISI][Medline]
  51. Wang JY, McCormack SA, Viar MJ, Wang H, Tzen CY, Scott RE, Johnson LR. Decreased expression of protooncogenes c-fos, c-myc and c-jun following polyamine depletion in IEC-6 cells. Am J Physiol Gastrointest Liver Physiol 265: G331–G338, 1993.[Abstract/Free Full Text]
  52. Wild GE, Murray D. Alteration in quantitative distribution of Na,K-ATPase activity along crypt-villus axis in animal model of malabsorption characterized by hyperproliferative crypt cytokinetics. Dig Dis Sci 37: 417–425, 1992.[CrossRef][ISI][Medline]
  53. Wild GE, Daly AS, Sauriol N, Bennett G. Effect of exogenously administered polyamine on the structural maturation and enzyme ontogeny of the postnatal rat intestine. Biol Neonate 63: 246–257, 1993.[ISI][Medline]
  54. Wild GE, Thomson ABR. Na+-K+-ATPase {alpha}1 and beta1 mRNA and protein levels in rat small intestine in experimental ileitis. Am J Physiol Gastrointest Liver Physiol 269: G666–G675, 1995.[Abstract/Free Full Text]
  55. Wild GE, Turner R, Chao LGS, Faria J, Keelan M, Clandinin MT, Thomson ABR. Dietary lipid modulation of Na+/glucose co-transporter (SGLT1), Na+,K+-ATPase, ornithine decarboxylase gene expression in the rat small intestine in diabetes mellitus. J Nutr Biochem 8: 673–680, 1997.[CrossRef][ISI]
  56. Wild GE, Thompson JA, Searles L, Turner R, Hasan J, Thomson ABR. Small intestinal Na+,K+-adenosine triphosphatase activity and gene expression in experimental diabetes mellitus. Dig Dis Sci 44: 407–414, 1999.[CrossRef][ISI][Medline]
  57. Zemelman BV, Walker WA, Chu SW. Expression and developmental regulation of Na+,K+-adenosine triphosphatase in the rat small intestine. J Clin Invest 90: 1016–1022, 1992.[ISI][Medline]




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