GLUT-5 expression in neonatal rats: crypt-villus location and age-dependent regulation

Lan Jiang1, Elmer S. David2, Noel Espina3, and Ronaldo P. Ferraris4

1 Graduate School of the Biomedical Sciences and Departments of 2 Pediatrics, 3 Anatomy, Cell Biology, and Injury Sciences, and 4 Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, New Jersey 07103-2714


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The rat fructose transporter normally appears after completion of weaning but can be precociously induced by early feeding of a high-fructose diet. In this study, the crypt-villus site, the metabolic nature of the signal, and the age dependence of induction were determined. In weaning rats fed high-glucose pellets, GLUT-5 mRNA expression was modest, localized mainly in the upper three-fourths of the villus, and there was little expression in the villus base. When fed high-fructose pellets, GLUT-5 mRNA expression was two to three times greater in all regions except the villus base. Intestinal perfusion in vivo of a nonmetabolizable fructose analog, 3-O-methylfructose, tended to increase fructose uptake rate and moderately increased GLUT-5 mRNA abundance but had no effect on glucose uptake rates and SGLT1 mRNA abundance. Gavage feeding of high-fructose, but not high-glucose, solutions enhanced fructose uptake only in pups >= 14 days, suggesting that GLUT-5 regulation is markedly age dependent. Fructose or its metabolites upregulate GLUT-5 expression in all enterocytes, except those in the crypt and villus base and in pups <14 days old.

membranes; metabolism; sugars; transport; small intestine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GLUT-5 IS THE FRUCTOSE TRANSPORTER of the small intestine (25). During rat development, the expression of GLUT-5 is considerably delayed compared with that of the Na+-dependent glucose transporter (SGLT1). In rats or rabbits, SGLT1 shows strong expression at birth, whereas GLUT-5 is expressed only after weaning is completed (5, 25, 27, 29). However, fructose transporter activity and GLUT-5 mRNA abundance in midweaning rats can be enhanced ahead of this natural schedule by precocious consumption of dietary fructose (10, 27). There is a different type of pattern in adult rats as their intestines already express GLUT-5 in significant amounts and GLUT-5 expression is subject to diurnal rhythms. Fructose feeding in adult rats increases GLUT-5 mRNA and protein levels threefold above the diurnal levels of expression (3, 8). In contrast, GLUT-5 expression in neonatal rats is not subject to diurnal rhythms (28). Reprogramming GLUT-5 development in midweaning rat pups requires de novo mRNA and protein synthesis (18), occurs independently of the well-known corticosterone and thyroxine surge that coincides with weaning (23), and relies solely on luminal signals, most likely, luminal fructose (28).

In this study, we address three topics that will increase our understanding of the mechanisms underlying the precocious modulation of GLUT-5 by dietary fructose in particular and of the interactions occurring between exogenous signals and endogenous factors regulating transporter development in general. The crypt-villus site of fructose transporter induction by dietary fructose during early development is not known. In adult rat and human small intestine, GLUT-5 mRNA is localized mainly in the midvillus region (25). In adult rats, epithelial location is not affected by time of day or diet (7), suggesting that circadian- or diet-induced changes in GLUT-5 mRNA abundance always occur in cells along the lower and midvillus regions.

There has also been no recent study on the role of fructose metabolism in the expression of the intestinal fructose transporter. After fructose is absorbed by the small intestine, some of it is metabolized, in part to lactate and glucose (17). Intravenous infusion of 3-O-methylfructose, a nonmetabolizable analog of fructose (17), led to increases in in vitro mucosal-to-serosal fluxes of 3-O-methylfructose and fructose in the adult rat small intestine (9), fluxes that were not inhibited by either phloretin, an inhibitor of GLUT-2 (20), or phlorizin, an inhibitor of SGLT1 (13). Although these results suggested that GLUT-5 can transport nonmetabolizable fructose analogs and that GLUT-5 expression can be regulated by nonmetabolizable fructose analogs in plasma (9), the study could not distinguish whether GLUT-5 induction is due to the transport of fructose itself or to subsequent metabolism of fructose in the cytosol.

Finally, although intestinal nutrient transport is clearly developmentally regulated (1, 2, 11), it is not known whether dietary or substrate regulation of nutrient transport itself is developmentally modulated. In the only study exploring this question, Toloza and Diamond (30) found that tadpoles, but not adult frogs, can regulate intestinal nutrient transport. Hence, intestinal regulatory capacity is lost during amphibian metamorphosis. Although mammalian development is less abrupt, the mammalian small intestine nevertheless undergoes a marked transition at weaning when it needs to acquire the ability to synthesize different types or numbers of nutrient transporters to match changes in intestinal luminal substrate concentration that occur as the pups shift from milk to an omnivorous diet. Is the ability to regulate intestinal fructose transport present in suckling pups or is it acquired during the transition to weaning?

We fed weaning rats pelletized diets known to enhance GLUT-5 expression (27) and then determined crypt-villus location by in situ hybridization. We tested the hypothesis that in neonatal rat small intestine, all enterocytes lining the villus will synthesize GLUT-5 mRNA in response to a high-fructose (HF) diet. We then perfused 3-O-methylfructose solutions in the small intestine in vivo and determined GLUT-5 mRNA abundance and activity. Here, we tested the hypothesis that the enhancement of GLUT-5 expression by luminal fructose requires fructose metabolism. Finally, we gavaged fructose solutions into suckling and weaning pups to determine the age dependence of GLUT-5 regulation. We tested the hypothesis that age determines the ability of the intestines to regulate intestinal fructose transport.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Adult male and female Sprague-Dawley rats weighing ~200 g were purchased from Taconic (Germantown, NY). Rats were kept in the research animal facility (RAF) and allowed free access to water and chow (Purina Mills, Richmond, IN). Male and female rats were mated in the RAF. After the female rats became pregnant, they were separated from the male rats and carefully monitored until the pups were born. The time and date of birth were recorded; age at birth was considered day 0.

Determination of crypt-villus location by in situ hybridization. Midweaning (20-day old) rat pups were randomly divided into two groups (n = 4 for each group) fed 65% fructose or glucose pellets for 2 days (for composition of diets, see Ref. 10). Rats were killed, and their small intestines were immediately flushed with ice-cold Krebs-Ringer-bicarbonate solution, isolated, and fixed in 10% buffered formalin solution. The tissues were transferred to a 4% paraformaldehyde solution, dehydrated by ethanol, embedded in paraffin, cut into 8-µm sections, and mounted on slides (Superfrost Plus slide, Fisher Scientific). In situ hybridization was performed using a kit (in situ hybridization and detection system, GIBCO BRL, Gaithersburg, MD). The sequences of the oligonucleotide probes used in hybridization were as follows: antisense, 5'-GAAGATCTAGATGGTGGTGAGGA-3' (~1360-1382, accession no. L05195), and sense, 5'-GAGGAGGATGAGGCTGAGAAGG-3' (~765-786, accession no. L05195). The latter sequence (nucleotide number ~765-1382) is based on rat GLUT-5 cDNA, yet is also unique for GLUT-5 compared with the homologous sequence for other GLUT transporters (4). Signal intensity was quantified using Image Pro Plus version 3.0 (Media Cybernectic, Silver Spring, MD) by a viewer blinded to the identification of each slide. Each villus was arbitrarily divided into four regions: the lowest 25% represented the villus base, the lower middle 25% served as the lower midvillus region, the upper middle 25% served as the midvillus region, and the uppermost 25% represented the tip. Ten spots were randomly chosen from each region from every villus. Every spot (4 × 8 µm2, covering parts of several cells) contained 20 pixel readings. The average of these 20-pixel readings was the pixel density of this spot. The average pixel density from 10 spots was the pixel density of the region. The pixel density in the villus base of the villus from animals fed a high-glucose (HG) diet, which was hybridized with the sense probe, was considered the baseline density and assigned a value of 100%. All pixel densities from other sections were normalized to this baseline value. There were 10 villi chosen from each animal. Every slide contained four different tissues (one each from 2 HF-fed rats and 2 HG-fed rats). This was a semiquantitive approach to determine whether there were changes in the relative amount of mRNA generated in an experimental system where control and experimental tissues were on the same slide and subjected to exactly the same hybridization treatments and detection methods.

Competitive inhibition of fructose uptake by 3-O-methylfructose. To determine whether 3-O-methylfructose, a nonmetabolizable fructose analog, utilizes the same transport pathways as fructose, we did competition experiments. Using intestines of rat pups (22 day old) fed 65% fructose pellets for 2 days, the uptake rate of tracer fructose was measured in the presence of 50 mM mannitol, glucose, fructose, or 3-O-methylfructose (in Ringer solution). The fructose uptake rate in the incubation solution with 50 mM mannitol was designated as 100%, and other uptakes were normalized to that value. Additional experiments comparing inhibition of tracer fructose uptake by 3-O-methylfructose and fructose were done at 10 and 100 mM.

Intestinal perfusion of sugar solutions. In this experiment, 3-O-methylfructose was used to determine whether fructose metabolism is needed for the increase in GLUT-5 expression induced by luminal fructose. Because of potential complications arising from consumption of pellets containing a nonmetabolizable sugar (preliminary work resulted in diarrhea), we resorted to a perfusion method used previously (18). Midweaning (22 day old) rat pups were randomly divided into three groups (n = ~4-5/group), and their intestines were perfused with 100 mM fructose, 3-O-methylfructose, or glucose for 4 h as follows.

Rat pups were anesthetized by intraperitoneal injection of ketamine cocktail (20% ketamine and 12.5% xylazine in 0.9% NaCl; 2.5 ml/kg body wt), and the perfusion procedure described by Jiang and Ferraris (18) was followed. Briefly, the abdominal cavity was cut open, and the small intestine exposed. About 10 cm from the stomach, a small incision was made and a catheter was securely placed into the small intestine. Another catheter was inserted into the ileum 10 cm from the ileal-cecal valve. After flushing the chyme, the small intestine was continuously perfused with sugar solutions (60 ml/h at 37°C). Perfusion had no effect on intestinal and villous morphometry (18). The composition of the perfusion solution was (in mM) 78 NaCl, 4.7 KCl, 2.5 CaCl2 · H2O, 1.2 MgSO4, 19 NaHCO3, 2.2 KH2CO3, and 100 sugars. Four 1-cm jejunal segments were then isolated from each rat for determinations of glucose and fructose uptake rate. The tissue 15 to 20 cm from the pylorus was frozen in liquid nitrogen and stored in -80°C for later Northern blot analysis (28).

Effect of age on precocious enhancement of GLUT-5 expression. Pups of each age group (see Fig. 8) were divided into three groups: one group was gavaged (2% of body wt twice a day) with 65% fructose solution and another with 65% glucose solution, whereas a third (control) group remained with the dam and had access to mother's milk and/or chow (MMC). Gavage feeding was utilized as a method to upregulate GLUT-5, because suckling pups could not eat pelletized diets (in this case, they would have access only to mother's milk). Intestinal perfusion was possible for suckling pups but keeping them completely anesthetized and alive during perfusion proved difficult. Pups were returned to the dam after each gavage, hence, gavaged pups also had access to milk. Pups were gavaged for 2 consecutive days and killed on the third day. There were five age groups subjected to gavage feeding: 8-10 (i.e., gavage fed at 8 and 9 days of age and killed at 10 days of age), 10-12, 12-14, 14-16, and 20-22 days of age. The oldest age group was used as a positive control, because earlier studies (10, 23, 27) using 20-, 21-, or 22-day-old pups fed 65% fructose pellets for 2-5 days showed enhanced GLUT-5 expression.

Comparison of various feeding methods. Because we used three different methods to introduce fructose into the intestinal lumen, we compared the effect of each method on intestinal fructose transport (for each method, pups were 22 days old at the time of death). One group was fed pelletized diets, another was gavaged with sugar solutions, and a third was killed after the intestines were perfused for 4 h with sugar solutions. The pups fed pelletized diets for 2 days served as control to demonstrate previously observed enhancement of fructose transport in rats fed fructose pellets. Within each group, there were three subgroups: pups fed or perfused with fructose or glucose solutions or unperfused pups that remained with the dam. Litters were equally represented in each subgroup.

Northern blots. Total RNA was isolated by single-step RNA isolation. About 60 µg of total RNA were subjected to 1% agarose-6% formaldehyde electrophoresis (Horizon 11.14, Life Technologies, Gaithersburg, MD) and then transferred to a nitrocellulose membrane (Hybond-NX, 0.2 µm, Amersham) by capillary action. cDNA probes of rat GLUT-5, rat SGLT1, and mouse 18S (a control for loading and transfer) were each labeled with [32P]dCTP using a random primer labeling kit (RTS RadPrime DNA labeling system, GIBCO BRL). Hybridization of the nitrocellulose membrane to 32P-labeled cDNA was performed overnight in a solution of 50% deionized formamide 6× SSC (1× SSC is 0.15 M NaCl, 0.01 M NaHPO4, and 0.001 M EDTA, pH 7.4), 2.5× Denhardt's solution, 0.3%-0.5% SDS, and 100 µg/ml salmon sperm DNA at 42°C. The hybridized membrane was washed four times for 30 min each time with 0.1× SSC and 0.1% SDS at 60°C. Quantification of X-ray films was performed using a densitometry system (IS-1000 Digital Imaging System, Alpha Innotech). For probes, we used rat GLUT-5 cDNA (a gift from Dr. C. Burant), rat SGLT1 (generated by RT-PCR; Jiang and Ferraris), and rat 18S cDNA (a gift from Dr. M. Lee).

Sugar uptake measurements. Glucose and fructose uptake rates in the small intestine were determined following the technique of by Karasov and Diamond (19). Briefly, a 1-cm segment was everted and mounted on a grooved steel rod (3- mm diameter) and preincubated at 37°C for 5 min in Ringer solution bubbled with 95% O2-5% CO2. The sleeves were then incubated at 37°C in an oxygenated solution containing either D-[14C]glucose for 1 min or D-[14C]fructose for 2 min. L-[3H]glucose was used to correct for adherent fluid and passive diffusion of glucose or fructose. All radioisotopes were purchased from DuPont-NEN (Boston, MA). The solutions were stirred at 1,200 rpm during the incubation procedure to minimize unstirred layers. To reduce the radioactive label in the adherent fluid, there was a 20-s rinse in 30 ml of ice-old Ringer solution after incubation. The tissues were dissolved in a tissue solubilizer (Solvable, Packard Instruments). The dissolved tissue was mixed with scintillation cocktail (Ecolume, ICN), and radioactivity was measured with a liquid scintillation counter (Beckman LS 7800, Beckman, Fullerton, CA). The uptake rates of both D-glucose and D-fructose were determined at 50 mM and expressed as nanomoles per milligram wet weight of small intestine.

Statistical analysis. For every experiment, the average value of the sugar uptake rate or transporter mRNA abundance from those rats perfused with glucose solution (or with vehicle injection in some of the experiments) was designated as 100%. All the actual values of the sugar uptake rates or transporter mRNA abundance were then recalculated by normalizing to this value. A two-way ANOVA was first used to determine the significance of the difference of relative absorption rates and relative mRNA abundance among groups with different treatments. If there was a significant difference, a one-way ANOVA or unpaired t-test was used to determine the particular effect that caused the difference. Statistical analysis was conducted using the Statview program (Abacus Concepts, Berkeley, CA).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Enhancement of fructose uptake by luminal fructose. Luminal fructose specifically and precociously enhances intestinal fructose transport in weaning rat small intestine (28). We now show that all three methods of introducing fructose into the lumen can each enhance fructose transport by two or more times (P < 0.001 by one-way ANOVA in each case) in the small intestine of midweaning pups. Like all previous work, pellet feeding of 65% fructose, but not 65% glucose, for 2 days specifically enhanced intestinal fructose transport (Fig. 1). Fructose uptake in 65% glucose-fed pups was similar to that of littermates staying with the dam (MMC). Gavage feeding a 65% fructose solution, but not a 65% glucose solution, also markedly enhanced fructose transport. Finally, in vivo perfusion of a 100 mM fructose solution, but not 100 mM glucose solution, into the intestinal lumen enhanced intestinal fructose transport. In the three various methods of introducing sugars into the intestinal lumen, intestinal glucose transport was not enhanced by fructose or glucose solutions or pellets (data not shown; Refs. 10, 23, 27), and intestinal fructose transport was not enhanced by glucose pellets or glucose solutions.


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Fig. 1.   The effect of diet and method of sugar entry into the intestinal lumen on relative fructose transport rate. Sugars were introduced into the gastrointestinal lumen of 22-day-old rat pups by consumption of 65% (wt/wt) sugar pellets for 2 days, continuous in vivo perfusion for 4 h of 100 mM sugar solutions in anesthetized rats, and gavage feeding of 65% (vol/vol) sugar solutions twice daily for 2 days. Bars are means ± SE; n = 5-8. In each group, fructose uptake rate in intestines from high-fructose diet (HF) rats or unperfused littermates with access to mother's milk and chow (MMC) was normalized to that in intestines from high-glucose diet (HG) pups, which was designated as 100%. The control uptake rates were 1.36 ± 0.15 nmol · min-1 · mg-1 for pellet-fed rats, 0.58 ± 0.10 nmol · min-1 · mg-1 for perfused rats, and 1.25 ± 0.15 nmol · min-1 · mg-1 for gavage-fed rats. * Significant difference between HF and HG. Fructose clearly enhanced fructose uptake rate in the intestine of weaning rats, regardless of the method used to introduce fructose into the lumen.

Crypt-villus location of GLUT-5 mRNA. There were marked effects of diet (P < 0.0001 by 2-way ANOVA) and crypt-villus region (P < 0.0001) on GLUT-5 mRNA localization (Figs. 2-4). In pups fed HG, there was a 1.6-2.8 greater (P < 0.01) abundance of GLUT-5 mRNA in the villus tip and middle compared with that in the villus base. Hence, there was a modest crypt-villus gradient of GLUT-5 message in pups fed HG.


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Fig. 2.   Representative photographs depicting by in situ hybridization GLUT-5 mRNA in the intestinal villi of pups fed HF (a) or HG pellets (b) then probed with antisense oligonucleotides or pups fed HF (c) or HG pellets (d) and probed with sense oligonucleotides. Blue regions in the cytosol indicate areas with relatively high concentrations of GLUT-5 mRNA. The in situ hybridization kit uses bovine alkaline phosphatase as the signaling system. The small intestine contains a sizable amount of alkaline phosphatase, particularly in the brush-border membrane of enterocytes (14), and this enzyme may nonspecifically react with the dye during the staining of sense and antisense slides. Efforts to block this nonspecific reaction by Levamisole, an alkaline phosphatase inhibitor, proved ineffective. Image analyses were done only in regions of cells at least 3 µm from the inner edge of the stained brush-border membrane (see MATERIALS AND METHODS for details). Scale bar = 60 µm.



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Fig. 3.   Representative photographs depicting by in situ hybridization GLUT-5 mRNA in the villus tip (a), upper villus (b), lower villus (c), and villus base (d) of intestines of rats fed HF pellets and probed with antisense oligonucleotide. The villus tips from rats fed HG pellets had lower levels of GLUT-5 mRNA when probed with antisense oligonucleotide (e). f: the villus tip from a rat fed HF but probed with a sense oligonucleotide. Blue regions depict areas with relatively high concentrations of GLUT-5 mRNA; arrows in a and e show some of these locations. Rectangle in b represents an area 4 × 8 µm2 (see MATERIALS AND METHODS for details). Scale bar = 10 µm.



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Fig. 4.   Effect of diet on crypt-villus location of GLUT-5 mRNA. Values are means ± SE; n = 40 villi from 4 animals. HF rats were fed 65% HF pellets for 2 days; HG rats were fed 65% HG pellets for 2 days. The pixel density in the villus base from animals fed the HG diet was designated as 100%. All other pixel densities were normalized to this value. Superscript letters indicate significant differences among crypt-villus regions within HF or HG. * Significant differences between HF and HG rats for the same villus region.

When pups were fed a HF diet, GLUT-5 mRNA abundance was markedly greater (~ 4-6 times greater, P < 0.01) along the middle and tip compared with the base regions of the villus. Hence, the crypt-villus gradient of GLUT-5 mRNA abundance in fructose-fed rats was much steeper than that in glucose-fed rats. There was no significant difference (P = 0.9) between HF and HG pups in GLUT-5 mRNA abundance of the villus base. Thus HF feeding increased GLUT-5 mRNA expression mainly in the upper three-fourths of the villus.

Inhibition of tracer fructose uptake by 3-O-methylfructose. Compared to unlabeled mannitol (50 mM), unlabeled fructose resulted in a 65% inhibition (P < 0.0001) of [14C]fructose uptake (Fig. 5). Unlabeled glucose or 3-O-methylfructose also inhibited [14C]fructose uptake but only by 30% (P <=  0.03). Another series of experiments produced similar results (not shown). At 10 mM, both 3-O-methylfructose and fructose did not significantly inhibit tracer fructose uptake; at 100 mM, 3-O-methylfructose inhibited tracer fructose uptake by 35%, whereas fructose inhibited uptake by 75%. Hence, 3-O-methylfructose is likely to be absorbed by GLUT-5, but it is a poor competitor of fructose uptake.


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Fig. 5.   The effect of addition of unlabeled sugars (50 mM) on mean relative uptake of tracer D-[14C]fructose by rat small intestine. The uptake rate (0.43 ± 0.03 nmol · min-1 · mg-1) in 50 mM mannitol (Man), a nontransportable, nonmetabolizable sugar alcohol, was designated as 100%. Uptake in the presence of other sugars [glucose (Glu), 3-O-methylfructose (3Omf), fructose (Fru)] was normalized to that value. Bars with different superscript letters are significantly different.

Effect of perfusion of 3-O-methylfructose on GLUT-5 expression. Fructose perfusion increased (P = 0.0001) GLUT-5 mRNA abundance by almost four times (Fig. 6, A and B). Perfusion with 3-O-methylfructose also significantly increased GLUT-5 mRNA abundance but only by two times more than glucose perfusion. Perfusion of sugar solutions had no effect (P = 0.49) on SGLT1 mRNA abundance (Fig. 6, C and D).


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Fig. 6.   The effect of HF, HG, and 3-O-methylfructose (3OMF) perfusion on GLUT-5 and SGLT1 mRNA abundance. A: representative Northern blot analysis of GLUT-5 mRNA. B: mean relative abundance of GLUT-5 mRNA. 18S is a loading and transfer control. GLUT-5 mRNA abundance was normalized to the density of 18S in the same blot. The ratio of GLUT-5 mRNA abundance to 18S rRNA abundance in HG-perfused intestines was then designated as 100%. The ratios in HF- and 3-O-methylfructose-perfused intestines were subsequently normalized to this value. Superscript letters indicate significant differences in relative GLUT-5 mRNA abundance among animals perfused with different sugar solutions. C: representative Northern blot analysis of SGLT1 mRNA. D: mean relative abundance of SGLT1 mRNA. The normalization procedure was similar to that in B. Perfusion with various sugars had no effect on SGLT1 mRNA abundance. B and D: values are means ± SE; n = 4.

Compared with glucose perfusion, fructose perfusion markedly increased fructose uptake rates (P <=  0.04, Fig. 7A). The effect of 3-O-methylfructose perfusion on fructose transport rate was modest and equivocal; it was at the same time similar to the effect of HF and HG perfusion. Glucose uptake rates were independent of perfusion solution (P = 0.27, Fig. 7B).


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Fig. 7.   The effect of HF, HG, and 3-O-methylfructose perfusion on fructose (A) and glucose uptake (B). Fructose uptake in HG pups equaled 0.37 ± 0.11 nmol · min-1 · mg-1 and was designated as 100%. Superscript letters indicate significant differences in fructose uptake rate among animals perfused with different sugar solutions. Glucose uptake of 100% equaled 3.65 ± 0.5 nmol · min-1 · mg-1. Perfusion with 3-O-methylfructose had no significant effect on fructose and glucose uptake. Values are means ± SE; n = 4.

Effect of age on enhancement of fructose transport. Intestinal fructose transport in the proximal (P = 0.83, Fig. 8) and middle intestines (P = 0.20, Fig. 9) was not enhanced by fructose gavaged into the intestine for two consecutive days before death in pups at age 10 days. In pups killed at 12 days of age, similar results were obtained: no enhancement of fructose transport in the proximal (P = 0.38) and middle intestine (P = 0.58). In pups killed at 14 days, however, there was a modest, diet-induced increase in fructose transport in the proximal (~1.5 times greater, P = 0.04) but not the middle small intestine (P 0.10). When pups were killed at 16 days of age, 2 consecutive days of fructose consumption by gavage markedly enhanced fructose transport in both proximal (~2.5 times greater, P = 0.0004) and middle intestines (~1.7 times greater, P = 0.03). Fructose transport was also enhanced in the proximal small intestine of a control, fructose-gavaged age group (22 days), although the HF-induced increase in the middle intestine did not reach statistical significance. However, fructose transport was enhanced in both proximal and middle small intestines of 22-day-old pups fed HF in previous experiments (10, 23, 27). In all age groups, fructose transport in the distal small intestine was unaffected by fructose solutions fed by gavage (not shown). Intestinal glucose transport was independent of diet in all age groups (P >=  0.24, Figs. 8 and 9).


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Fig. 8.   Fructose (A) and glucose uptake (B) in the proximal small intestine of rats of various age groups. HF or HG solutions were fed by gavage to pups twice a day for 2 days before death at ages 10, 12, 14, 16, and 22 days. Uptake in HF and MMC pups was normalized to that in HG pups designated as 100% for each age group. Gavage feeding with HF enhanced fructose uptake, but only in pups aged 14 days or older. For unknown reasons, HF also had an effect on glucose uptake at 14 days of age. Gavage feeding with HG had no effect on fructose and glucose uptake. Values are means ± SE; n = 4-8. * Significant difference.



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Fig. 9.   Fructose (A) and glucose (B) uptake in the middle small intestine of rats of various age groups. Normalization procedure is the same as in Fig. 8 legend. Gavage feeding with HF enhanced fructose uptake by the middle small intestine but only in 16-day-old pups. Gavage feeding with HG had no effect on fructose and glucose uptake. Values are means ± SE; n = 4-8. * Significant difference.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Dietary fructose enhances GLUT-5 mRNA expression in villus enterocytes. In early weaning rats fed glucose, GLUT-5 mRNA is expressed at a low level throughout the crypt-villus axis, a finding consistent with previous Northern blot results indicating low GLUT-5 mRNA expression in rats fed glucose (27). The two- to threefold increase in GLUT-5 mRNA detected by in situ hybridization is also consistent with the two- to threefold increase in mean GLUT-5 mRNA abundance measured by Northern blot analysis of total RNA from pups fed HF pellets (27) and pups perfused with HF solutions (18).

In adult rats fed chow, GLUT-5 mRNA abundance analyzed by Northern blotting was highest in enterocytes collected by cell fractionation from the upper and middle villus regions (3). Fructose feeding greatly enhanced GLUT-5 mRNA abundance along the villus, with the greatest increase occurring in the tips of the villus. This result is consistent with what we found in the weaning rat small intestine by in situ hybridization. In contrast, Corpe et al. (7) found by in situ hybridization that GLUT-5 mRNA was almost nonexistent in the crypt, but GLUT-5 mRNA abundance was highest in the lower to middle villus regions in adult rats. Gavage feeding of fructose solution for 4 h increased GLUT-5 mRNA abundance but did not alter the crypt-villus pattern of distribution (7). Although there are differences in observations of the distribution of GLUT-5 message in the villus, two common findings among these studies are that GLUT-5 mRNA abundance in villus cells increases with fructose feeding and GLUT-5 mRNA is virtually absent in the crypts. Corpe et al. (7) suggested that crypt cells are unable to sense and respond to dietary fructose. It is also possible that fructose is sensed by crypt cells, but these cells are unable to respond until they have migrated into the villus regions. In the adult mouse, adaptive upregulation of SGLT1 by diet indicates that crypt cells sense the carbohydrate signal from the lumen (12).

The cell transit time from the crypt to the villus tip in intact rats is 2 days for 22-day-old rats (31), and this finding confounds the interpretation of results from our in situ experiments in which midweaning (20-day old) pups were fed HF pellets for ~ 40 h. Hence, it will not be possible to clearly determine the crypt-villus site of GLUT-5 induction. However, we have every reason to believe that the crypt-villus pattern of expression observed in intestines of pups fed fructose pellets for 40 h (Fig. 4) would be the same in intestines perfused with fructose solutions for 4 h (Fig. 1). Moreover, we have previously observed parallel increases in intestinal GLUT-5 mRNA abundance after 4 h of fructose perfusion and 4 h after initial consumption of fructose pellets (18). Even with a fast migration rate that can lead to cell turnover times of 2 to 3 days, effects within 4 h would indicate that it is the villus cells that are responding.

In contrast to the circadian regulation of sugar transporters in the small intestine of adult rats (5, 8, 26), GLUT-5 mRNA expression and transporter activity in weaning rats do not follow a circadian rhythm (27). Differences in circadian control might be due to the differences in feeding habits between adult and weaning rats (16). Neonatal rats consume food whenever they are hungry, regardless of time of day.

3-O-methylfructose is a poor substrate of GLUT-5. When rat GLUT-5 was expressed in Xenopus oocytes, [14C]fructose uptake was inhibited by ~75% and 40% in the presence of unlabeled fructose (50 mM) and glucose, respectively (25), a finding similar to that shown in Fig. 5. The failure of unlabeled fructose to completely inhibit tracer fructose uptake is puzzling, but it is possible that the concentration of unlabeled fructose was not high enough to completely inhibit the uptake of labeled fructose. However, increasing the unlabeled fructose concentration by two times to 100 mM did not significantly increase inhibition of tracer uptake. Further increases in unlabeled fructose concentration were not attempted because they would have resulted in ionic imbalances and confounded results.

Because glucose partially inhibits tracer fructose uptake, either GLUT-5 transports some glucose or glucose is inhibiting the component of tracer fructose uptake that is not mediated by GLUT-5. Recent work (15) suggests that GLUT-2, the monosaccharide transporter thought to reside exclusively in the basolateral membrane (6), may also be found in the brush-border membrane.

The methyl group in 3-O-methylfructose makes the molecule bulky and hydrophobic and may decrease its affinity for GLUT-5 compared with that of fructose. Hence, 3-O-methylfructose inhibits tracer fructose uptake two to three times less efficiently than unlabeled fructose. Nevertheless, results suggest that 3-O-methylfructose is likely to be absorbed by intestinal cells and to be a relatively low-affinity substrate of GLUT-5.

Potential role of fructose metabolism. Fructose can be metabolized in the adult rat small intestine, and activities of intestinal enzymes required for fructose metabolism can also be enhanced after consumption of a HF diet (21, 22). Although the rat small intestine can metabolize fructose, most of the fructose absorbed by the small intestine is transported to the liver through the portal vein (22). Fructose metabolism in the liver does not seem to be involved in the upregulation of GLUT-5 expression, because HF feeding does not affect the fructose uptake rate and GLUT-5 mRNA abundance in a bypassed loop of small intestine with intact blood supply and nerve connections (3, 28). Thus the upregulation of the intestinal fructose transporter by dietary fructose is not due to systemic signals; otherwise fructose-derived metabolites from the liver would also have enhanced GLUT-5 expression in the bypassed loop.

The signal to upregulate GLUT-5 can either be intracellular fructose or its metabolites. If GLUT-5 expression and activity are equally enhanced by perfusion of 3-O-methylfructose or fructose, then fructose metabolism will not be required for upregulation. Conversely, if 3-O-methylfructose did not enhance GLUT-5 expression, there may be a potential role for fructose metabolites. Unfortunately, perfusion of 3-O-methylfructose increased GLUT-5 mRNA by only two times and had no significant effect on fructose uptake, equivocal results that do not support either of the two hypotheses. Interpretation of these results is also complicated by differences in the apparent affinity of GLUT-5 for fructose and 3-O-methylfructose. Hence, the data with 3-O-methylfructose are inconclusive.

Age-dependent modulation of fructose transport. Fructose transport can apparently only be enhanced in weaning but not suckling rats, indicating that dietary modulation of fructose transport is age dependent. Suckling rats are completely dependent on dam's milk for their nutrition and cannot possibly survive without the dam because their teeth are not yet developed. The composition of milk is constant, and therefore there is no need to regulate the activity of nutrient transporters in the small intestine of suckling rats. As rats wean, they gradually shift to an omnivorous diet, and the regulatory system for GLUT-5 develops in parallel with the dietary shift. There are three phases of GLUT-5 development in the small intestine: in suckling rats, GLUT-5 cannot be modulated by luminal fructose; in weaning rats, GLUT-5 expression can be precociously induced if luminal fructose is introduced; and in adult rats, modest GLUT-5 expression and activity can be enhanced by dietary fructose.

Regulatory systems have also been found to be age dependent in other systems. In frogs, glucose and proline transport can only be modulated by diet in the tadpole stage and not in adults (30). In rats, exogenous corticosterone modulates sucrase isomaltase activity only between 10 and 16 days of age and not at 18 days or older (24). Clearly, additional study is needed to identify age-sensitive regulatory factors that modulate GLUT-5 expression.


    ACKNOWLEDGEMENTS

We thank Drs. C. Burant and M. Lee for the cDNA probes and Drs. G. Diamond, F. Diecke, N. Ingoglia, I. Monteiro, and P. Rameshwar for valuable discussion.


    FOOTNOTES

This work was supported by National Science Foundation Grant IBN-9985808, National Institute on Aging Grant AG-11403, and United States Department of Agriculture and Northeastern Regional Aquaculture Center Grant 94-38500-0044.

This work was submitted by L. Jiang in partial fulfillment of requirements for a doctorate in biomedical sciences.

Address for reprint requests and other correspondence: R. P. Ferraris, Dept. of Pharmacology and Physiology, Univ. of Medicine and Dentistry of New Jersey-New Jersey Medical School, 185 S. Orange Ave., Newark, NJ 07103-2714 (E-mail: ferraris{at}umdnj.edu).

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.

Received 16 December 2000; accepted in final form 3 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Buddington, RK, and Diamond JM. Ontogenetic development of intestinal nutrient transporters. Annu Rev Physiol 51: 601-619, 1989[ISI][Medline].

2.   Buddington, RK, and Malo C. Intestinal brush-border membrane enzyme activities and transport functions during prenatal development of pigs. J Pediatr Gastroenterol Nutr 23: 51-64, 1996[ISI][Medline].

3.   Burant, CF, and Saxena M. Rapid reversible substrate regulation of fructose transporter expression in rat small intestine and kidney. Am J Physiol Gastrointest Liver Physiol 267: G71-G79, 1994[Abstract/Free Full Text].

4.   Casirola, DM, Lan Y, and Ferraris RP. Effects of changes in calorie intake on intestinal nutrient uptake and transporter mRNA levels in aged mice. J Gerontol A Biol Sci Med Sci 52: B300-B310, 1997[Abstract].

5.   Castello, A, Guma A, Sevilla L, Furriols M, Testar X, Palacin M, and Zorzano A. Regulation of GLUT5 gene expression in rat intestinal mucosa: regional distribution, circadian rhythm, perinatal development and effect of diabetes. Biochem J 309: 271-277, 1995[ISI][Medline].

6.   Cheeseman, CI. GLUT2 is the transporter for fructose across the rat intestinal basolateral membrane. Gastroenterology 105: 1050-1056, 1993[ISI][Medline].

7.   Corpe, CP, Bovelander FJ, Hoekstra JH, and Burant CF. The small intestinal fructose transporters: site of dietary perception and evidence for diurnal and fructose sensitive control elements. Biochim Biophys Acta 1402: 229-238, 1998[ISI][Medline].

8.   Corpe, CP, and Burant CF. Hexose transporter expression in rat small intestine: effect of diet on diurnal variations. Am J Physiol Gastrointest Liver Physiol 271: G211-G216, 1996[Abstract/Free Full Text].

9.   Csaky, TZ, and Fischer E. Effects of ketohexosemia on the ketohexose transport in the small intestine of rats. Biochim Biophys Acta 772: 259-263, 1984[ISI][Medline].

10.   David, ES, Cingari DS, and Ferraris RP. Dietary induction of intestinal fructose absorption in weaning rats. Pediatr Res 37: 777-782, 1995[Abstract].

11.   Ferraris, RP, and Diamond J. Regulation of intestinal sugar transport. Physiol Rev 77: 257-302, 1997[Abstract/Free Full Text].

12.   Ferraris, RP, and Diamond JM. Crypt/villus site of substrate-dependent regulation of mouse intestinal glucose transporters. Proc Natl Acad Sci USA 90: 5868-5872, 1993[Abstract].

13.   Ferraris, RP, and Diamond JM. Use of phlorizin binding to demonstrate induction of intestinal glucose transporters. J Membr Biol 94: 77-82, 1986[ISI][Medline].

14.   Ferraris, RP, Villenas SA, and Diamond J. Regulation of brush-border enzyme activities and enterocyte migration rates in mouse small intestine. Am J Physiol Gastrointest Liver Physiol 262: G1047-G1059, 1992[Abstract/Free Full Text].

15.   Helliwell, PA, Richardson M, Affleck J, and Kellett GL. Stimulation of fructose transport across the intestinal brush-border membrane by PMA is mediated by GLUT2 and dynamically regulated by protein kinase C. Biochem J 350: 149-154, 2000[ISI][Medline].

16.   Henning, SJ. Functional development of gastrointestinal tract. In: Physiology of the Gastrointestinal Tract (2nd ed.), edited by Johnson LR.. New York: Raven, 1987, p. 285-300.

17.   Holloway, PA, and Parsons DS. Absorption and metabolism of fructose by rat jejunum. Biochem J 222: 57-64, 1984[ISI][Medline].

18.   Jiang, L, and Ferraris RP. Developmental reprogramming of rat GLUT-5 requires de novo mRNA and protein synthesis. Am J Physiol Gastrointest Liver Physiol 280: G113-G120, 2001[Abstract/Free Full Text].

19.   Karasov, WH, and Diamond JM. Adaptive regulation of sugar and amino acid transport by vertebrate intestine. Am J Physiol Gastrointest Liver Physiol 245: G443-G462, 1983[Abstract/Free Full Text].

20.   Kellett, GL, and Helliwell PA. The diffusive component of intestinal glucose absorption is mediated by the glucose-induced recruitment of GLUT2 to the brush-border membrane. Biochem J 350: 155-162, 2000[ISI][Medline].

21.   Korieh, A, and Crouzoulon G. Dietary regulation of fructose metabolism in the intestine and in the liver of the rat. Duration of the effects of a high fructose diet after the return to the standard diet. Arch Int Physiol Biochim Biophys 99: 455-460, 1991[ISI][Medline].

22.   Mayes, PA. Intermediary metabolism of fructose. Am J Clin Nutr 58 Suppl: 754S-765S, 1993[Abstract].

23.   Monteiro, IM, and Ferraris RP. Precocious enhancement of intestinal fructose uptake by diet in adrenalectomized rat pups. Pediatr Res 41: 353-358, 1997[Abstract].

24.   Nanthakumar, NN, and 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].

25.   Rand, EB, Depaoli AM, Davidson NO, Bell GI, and Burant CF. Sequence, tissue distribution, and functional characterization of the rat fructose transporter GLUT5. Am J Physiol Gastrointest Liver Physiol 264: G1169-G1176, 1993[Abstract/Free Full Text].

26.   Rhoads, DB, Rosenbaum DH, Unsal H, Isselbacher KJ, and Levitsky LL. Circadian periodicity of intestinal Na+/glucose cotransporter 1 mRNA levels is transcriptionally regulated. J Biol Chem 273: 9510-9516, 1998[Abstract/Free Full Text].

27.   Shu, R, David ES, and 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].

28.   Shu, R, David ES, and Ferraris RP. Luminal fructose modulates fructose transport and GLUT-5 expression in small intestine of weaning rats. Am J Physiol Gastrointest Liver Physiol 274: G232-G239, 1998[Abstract/Free Full Text].

29.   Toloza, EM, and Diamond J. Ontogenetic development of nutrient transporters in rat intestine. Am J Physiol Gastrointest Liver Physiol 263: G593-G604, 1992[Abstract/Free Full Text].

30.   Toloza, EM, and Diamond JM. Ontogenetic development of transporter regulation in bullfrog intestine. Am J Physiol Gastrointest Liver Physiol 258: G770-G773, 1990[Abstract/Free Full Text].

31.   Yeh, KY. Cell kinetics in the small intestine of suckling rats. I. Influence of hypophysectomy. Anat Rec 188: 69-76, 1977[ISI][Medline].


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