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Department of Pharmacology and Physiology (V.D., X.-L.C., R.P.F.) and Center for Applied Genomics (P.S.), Public Health Research Institute, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07101-1709
Address all correspondence and requests for reprints to: R. P. Ferraris, Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07101-1709. E-mail: ferraris{at}umdnj.edu.
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Abstract |
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Introduction |
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GLUT5 is localized in the apical membrane of intestinal cells (9), and cytosolic fructose is transported to the blood by the basolateral glucose/fructose transporter GLUT2 (Slc2A2), which has also been hypothesized to participate in the apical transport of sugars (10). The third major sugar transporter is the sodium-glucose cotransporter 1 (SGLT1) (Slc5A1), located in the apical membrane. Recently, another glucose/fructose transporter, GLUT7 (Slc2A7), had been identified (11) in the apical membrane. However, only GLUT5 is specifically and markedly regulated by fructose (12); in fact, its response is directly proportional to luminal concentrations of fructose. In contrast, GLUT5 expression in intestines perfused with glucose is always low and the same as those in intestines perfused with nonmetabolizable glucose or fructose analogs (6). In weaning, 20-d-old rat intestines characterized by fructose sensitivity, we have already identified by microarray analysis 50 other fructose-responsive genes, paralleling the changes in GLUT5 expression (13).
In this study we first tested, using microarray hybridization, the hypothesis that the population of fructose-responsive genes at 20 d of age is different from the population of fructose-responsive genes in the 10-d-old pups when GLUT5 is fructose insensitive. For this hypothesis, intestines of same-age (10 d old) pups were compared after either fructose or glucose perfusion. Having proven this hypothesis, we then tried to identify regulatory genes that modulate fructose sensitivity by tracking changes in expression as a function of age. For this, the intestines of 10- and 20-d-old pups perfused with fructose were compared by microarray analysis (same perfusion solution, different age groups). Intestines of 10- and 20-d-old pups perfused with glucose were used to segregate age- but not fructose-responsive genes. When the microarray results revealed that a significant number of age- and fructose-responsive genes was modulated by glucocorticoids, we then tested the hypothesis that corticosteroids play a major role in regulating intestinal GLUT5 development by injecting pups with a synthetic glucocorticoid, dexamethasone (dex).
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Materials and Methods |
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). At 20 d old, five pups were perfused with the glucose (20G) and five with the fructose solution (20F). Glucose and fructose solutions were continuously perfused for 4 h; we have previously shown that 4-h perfusion is sufficient time to detect fructose-induced changes in GLUT5 mRNA abundance, GLUT5 protein abundance in the apical membrane, and in fructose transport rate (7). The sugar concentration (100 mM in Ringer solution) was also based on previous work indicating that peak luminal sugar concentration in rats fed high carbohydrate (14) exceeded 100 mM.
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). In the first set, the intestinal gene expression levels from 10-d-old rats perfused with glucose were compared with those perfused with fructose (10G vs. 10F, respectively) by microarray to determine fructose-responsive genes in the suckling stage. These genes are fructose responsive but age independent. With a second set of microarrays, we identified fructose-responsive genes whose expression also changes with age. For this, we compared the expression of the genes in the intestines of 10 and 20-d-old pups perfused with fructose (10 vs. 20F, respectively), to yield a list of genes that change with age under fructose perfusion. We also compared the expression of genes in the intestines of 10- and 20-d-old pups perfused with glucose. This comparison between 10- and 20-d-old pups perfused with glucose was used as control to identify genes that are not fructose responsive but whose expression changes with age.
Experiment 2: dex study.
To determine the role of glucocorticoids in GLUT5 development, we examined the effect of dex on fructose induction of GLUT5 in the 10-d-old pups. Pups received daily injections for 2-d dex (ip 50 ng/kg body weight) or vehicle (the same volume of 5% dimethyl sulfoxide, the solvent for dex, in saline solution) beginning at 8 d of age. dex and vehicle-treated animals were perfused at 10 d of age with glucose or fructose solution as described previously. The dose and time course of dex injection were chosen because preliminary work showed that a daily injection of dex for 2 d, from 40–400 ng/kg body weight (the dose for humans), is effective in inducing GLUT5 expression in the small intestine of 10-d-old fructose-perfused pups.
Animals
All the procedures conducted in this study were approved by the Institutional Animal Care and Use Committee, University of Medicine and Dentistry of New Jersey, New Jersey Medical School. Pregnant female Sprague Dawley rats purchased from Taconic (Germantown, NY) were housed in the research animal facility under a 12-h light, 12-h dark photoperiod in a temperature-controlled room (22–24 C). Dams were fed ad libitum a commercial diet (Purina Mills, Richmond, IN). After birth, rat pups were kept with their dams; age at birth was considered d 0. At 10 or 20 d old, rat pups were removed randomly from their dams and used for perfusion. The pups used for the dex study were injected once daily for 2 d beginning at 8 d of age with dex or vehicle and then removed from the dam at 10 d of age for perfusion experiments.
Intestinal perfusion
The rat intestinal perfusion procedure was conducted following the method previously described (7). Rat pups (10 and 20 d old, not starved) were initially anesthetized (0.2–0.4 ml/100 g body weight, ip, of ketamine 20 mg/ml and xylazine 2.5 mg/ml) and kept under continuous anesthesia for 4-h perfusion. Then after opening the abdominal cavity, the intestine was exposed, and a small incision was made 5-cm distal to the ligament of Treitz and 10-cm proximal from the ileocecal valve, and a catheter was inserted into the lumen. After the contents were flushed, the small intestine was continuously perfused with sugar solution (100 mM fructose or glucose in Ringer) at a rate of 30 ml/h at 37 C using a peristaltic pump. Composition of the perfusion solution was as follows (in mM): 78 NaCl, 4.7 KCl, 2.5 CaCl2 · 5H2O, 1.2 MgSO4, 19 NaHCO3, 2.2 KH2PO4, and 100 glucose or fructose (pH 7.4) (300 mOsm). After perfusion, sugar uptakes were measured in vitro, and tissues for mRNA analysis were frozen.
Glucose and fructose uptakes
Briefly, four 1-cm segments were everted and mounted on grooved steel rods and preincubated at 37 C for 5 min in Krebs ringer bicarbonate buffer [in mM: NaCl 128, KCl 4.7, NaHCO3 19, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2 (pH 7.4)] bubbled with 95% O2-5% CO2 (15). Two sleeves were then incubated under agitation (1200 rpm) 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. The tissues were quickly rinsed (20 sec) and processed for radioactivity as previously described. 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.
mRNA extraction, DNase treatment, and RT reaction
Total RNA was extracted from 100 mg scraped mucosa using 1 ml TRIZOL reagent (Invitrogen, Carlsbad, CA). The total RNA concentration was determined by spectrophotometry (Beckman DUR640; Beckman Coulter, Inc., Fullerton, CA) and the quality analyzed by 1% agarose gel electrophoresis with ethidium bromide staining. To hydrolyze contaminating DNA in the RNA preparations, 20 µg RNA was combined with 1 µl RQ1 Rnase free DNase I (Promega, Madison, WI) and 10 µl 10xDNase buffer in a final volume of 100 µl. After Dnase treatment, RNA concentration and quality were analyzed as described previously. The cDNA was generated from 2.5 µg DNase-treated RNA using SuperScript III RNase H – Reverse Transcriptase and oligo (dT)20 (Invitrogen) in a total volume of 20 µl.
Microarray analysis
The design, analysis, and interpretation of this microarray experiment followed the same protocol used previously by us (13). We used a rat 5000-oligonucleotide array built from 4854 oligonucleotides representing 4803 independent genes, and all oligonucleotides were 60–70 nucleotides in length [Rat 5K Oligo Array (NGEL 2.0.1); Center for Applied Genomics, Public Health Research Institute, Newark, NJ]. A detailed description of array design elements is available from the following web site: http://www.cag.icph.org. The cDNA was synthesized with the 3DNA Submicro Oligo Expression Array Detection Kit (Genisphere, Hatfield, PA) and labeled with fluorescent dyes Cy3 or Cy5 following the manufacturer’s instructions (13). In the microarray experiments, 10 slides were used to compare the 10-d-old rat intestines perfused with fructose (10F) (n = 5) against those perfused with glucose (10G) (n = 5), with each pair being littermates. cDNA from 10F was then labeled with Cy5, and cDNA from 10G with Cy3. To eliminate dye bias, 10F cDNA from pups in the next experiment was labeled with Cy3, whereas 10G cDNA was labeled with Cy5. Data from this dye flip were used to correct results as described later in Data processing. Moreover, two pools of the same amount of total RNA from each of five 10F and 10G pups were made. The 10F and 10G pools were self-against-self-hybridized on two additional slides to identify potentially false-positive genes.
In the second set, 10 microarray slides were used to compare the 10-d-old rat intestines perfused with fructose for 4 h (10F) (n = 5) against 20-d-old rat intestines also perfused with fructose (20F) (n = 5). cDNA from 10F intestines was labeled alternatively with Cy5 or Cy3, whereas the paired 20F cDNAs were labeled correspondingly with Cy3 or Cy5. This random Cy3 and Cy5 labeling switch between 10 and 20F, as well as pooled self-against self-hybridizations were used to eliminate dye bias. The same procedure was used for 10- and 20-d-old rats whose intestines were perfused with glucose (10 vs. 20G comparisons).
Data processing
The data processing, normalization, and statistical analyses were done as described by previous work (13). The results (median pixel intensity of each array spot) were normalized by the locally weighed scatter smoother method (16). To eliminate genes that were considered false positives and to choose genes that might change significantly with fructose perfusion or/and age, we followed the following criteria. First, genes whose expression was so low that their average median intensity was less than 2-fold that of background intensity were eliminated. Moreover, genes whose expression changed by more than 50% in self-self hybridization experiments were also eliminated. Then, sugar-induced changes in gene expression should not be altered when a different dye was used to label the same sample, so any gene whose expression changed from a positive value to a negative value when the dye was switched was eliminated. Finally, only genes that changed by more than or equal to 50% in at least four of five comparisons in the experiment involving 10 and 20-d-old pups, and in three of five comparisons in the experiment comparing 10-d-old pups perfused in fructose and glucose, were considered. We then performed a one-sample t test to determine significance of changes in gene expression in the microarray results, following earlier work (13). Gene expression was further analyzed using Pathway Assist (Stratagene, La Jolla, CA) and Bibliosphere (Genomatix Software Inc., Ann Arbor, MI), software applications that can build and examine biological association networks, including traditional pathways, among genes selected as nodes (see Fig. 3) to reveal gene regulatory networks.
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), and were purchased from Integrated DNA Technologies (IDT, Coralville, IA). Quantitative PCR analyses (MX 3000P; Stratagene) for candidate responsive genes were performed using the iTaq SYBR Green Supermix with Rox (Bio-Rad Laboratories, Hercules, CA) as described by Kirchner et al. (17). For each primer set (Table 1), the efficiency of the PCR reaction (linear equation: y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample (pool of reverse-transcribed RNA samples). Real-time PCR efficiencies (E) for each reaction were then calculated using the following equation: E = [10(1/slope)] – 1. Melting-curve analysis also was performed for each gene to check the specificity and identity of the RT-PCR products. The relative expression ratio of a target gene was calculated as follows:
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) of an unknown sample vs. a control (Ctcontrol – sample). In our experiment the control is the 10-d-old pups perfused with glucose solution. Because the expression level of elongation factor 1
(EF1) is not affected by F and G perfusion, or by age of pups (data not shown), the target gene expression has been normalized to EF1.
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Results |
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), and by 20- to 50-fold when compared with those in the intestines of 10-d-old pups. Fructose perfusion had no effect on the GLUT5 mRNA abundance in the intestine of the 10-d-olds pups. Compared with those in GLUT5, age-related increases in SGLT1 expression were very modest (approximately two times, Fig. 2B), and were significant only for glucose-perfused intestine. Larger glucose-induced increases in SGLT1 expression are usually observed in adult intestines (18). Intestinal GLUT2 mRNA abundance was independent of age and sugar perfusion (Fig. 2C).
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). Fructose perfusion in 20-d-old pups increased fructose uptake by 2.5 times compared with intestines of same-age pups whose intestines were perfused with glucose. In contrast, there was no effect of fructose perfusion on fructose uptake in the intestine of 10-d-old pups, whose fructose uptakes were similar to that in 20-d-old pups perfused with glucose. Glucose uptake rate increased modestly with glucose perfusion in 20-d-old pups (Fig. 2E).
Microarray results
Fructose-responsive genes in 10-d-old pups.
Using glucose-perfused intestines as control, we identified seven genes whose expression changes in the intestine of 10-d-old pups perfused with fructose for 4 h (Table 2). We found five genes that were significantly up-regulated between 1.5 and four times, and two down-regulated approximately 1.6 times with fructose perfusion. The up-regulation of glucose-6-phosphatase (G6Pc) has been confirmed by real-time PCR (2.3-fold; P < 0.05). As has previously been shown in Northern blots (6) and in Fig. 2A, there was no fructose-induced increase in intestinal GLUT5 mRNA abundance as measured by microarray at 10 d of age.
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). The complete list of genes was divided into three groups: those whose expression changed with age only under fructose perfusion (Tables 3 and 4); those whose expression changed only with age under glucose perfusion (supplemental Tables 1A and 2A, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org); and those whose expression changed with age under both fructose and glucose perfusion (Tables 5 and 6, and supplemental Tables 3A and 4A). DAVID and EASE bioinformatics resources were used to find the functional annotations [gene ontology (GO)], and the biological process of the differentially expressed genes (http://david.niaid.nih.gov/david/ease.htm).
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and 4, and Fig. 3). Some up- and down-regulated genes belonged to similar categories, primarily metabolism (nine are up-regulated genes and five down-regulated), transcription regulation (two up-regulated genes and three down-regulated), and signal transduction (three up-regulated and three down-regulated). It is quite interesting to note that development/proliferation/differentiation genes were mainly down-regulated with age, whereas receptor activity and hormone metabolism genes were mainly up-regulated with age. Two genes involved in steroid hormone metabolism, 17-β hydroxysteroid dehydrogenase type 2 (Hsd17b2) and 17-β hydroxysteroid dehydrogenase type 6 (Hsd17b6), were also up-regulated. The function of seven genes was not yet known. The fold change for up-regulated genes ranged between 1.6 (putative pheromone receptor) and 5.1 [retinol dehydrogenase type 2 (Rdh2)], whereas down-regulated genes exhibited fold changes between –5 [bleomycin hydrolase (Blmh)] and –1.56 [glucose-6-phosphate dehydrogenase (G6pdx)].
Genes differentially expressed between 20 and 10-d-old intestines perfused with glucose.
A total of 41 up-regulated and 46 down-regulated genes has been identified in the intestine of 20-d-old pups perfused only with glucose, relative to those in the 10-d-old pups (supplemental Tables 1A and 2A). The majority of the up-regulated genes belonged to three categories: metabolism (14 genes), cell-to-cell communication (14), and transport (7). The main categories of down-regulated genes were metabolism (10 genes), signal transduction (10), proliferation and development (15), and transport (5). Moreover, two genes down-regulated by age and glucose were involved in steroid hormone metabolism: Steroid 5--reductase 2 (Srd5a2) and 3β-hydroxysteroid dehydrogenase 1 (Hsd3b1). It is interesting to note that expression of glucose-sensitive genes, like that of fructose-sensitive genes, involved in proliferation and development was being markedly down-regulated as the rat was nearing completion of weaning.
Gene differentially expressed in 20- and 10-d-old intestines perfused with either fructose or glucose.
Relative to 10-d-old pups, we identified 71 genes up-regulated and 98 down-regulated in the intestine of 20-d-old pups perfused with either fructose or glucose. Therefore, these genes change in expression with age under either perfusion condition. In Tables 5 and 6 we show only the genes with more than 4-fold changes, the rest is presented in supplemental Tables 3A (for up-regulated) and 4A (down-regulated). The majority of these highly age-sensitive genes are involved in metabolism, transport, or development and differentiation activities (Fig. 4, A and B). The major difference between up- and down-regulated genes is that 28% of the down-regulated genes are involved in transduction activities, but only 6% in the up-regulated genes. Among the genes involved in carbohydrate metabolism, all of the up-regulated genes are involved in the glycolysis or Krebs cycle (enolase, pyruvate kinase, lactate dehydrogenase, phosphoglycerate kinase 1, and fumarate hydratase 1), whereas some down-regulated genes participate in gluconeogenesis (G6Pc and fructose-1,6- biphosphatase 1) (supplemental Tables 3A and 4A).
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Genes responsive to age and fructose, and those regulated by glucocorticoids
We reexamined by real-time PCR the expression of approximately 20 genes based on expression changes, GO analysis, and potential involvement in glucocorticoid metabolism or glucocorticoid sensitivity as identified using the Pathway Assist and Genomatix Bibliosphere programs (Table 6). We selected representative genes involved in the regulation of transcription and signal transduction to identify those potentially involved in GLUT5 regulation by age and fructose. The expression level of genes involved in carbohydrate metabolism (Table 6) or sugar transport (the results for GLUT5, SGLT1, and GLUT2 are illustrated in Fig. 2, A–C, respectively) were also measured by real-time RT-PCR. Finally, we analyzed eight genes identified as being glucocorticoid sensitive or involved in glucocorticoid/steroid metabolism.
By microarray analysis, the age-related changes in expression of some genes identified as transcription factors were either borderline or not significant, and these findings were in agreement with those obtained by real-time PCR (Table 6). The nuclear receptor for bile acids, nuclear receptor subfamily 1, group H, member 4 (Nr1h4), was confirmed to be down-regulated with age by two to three times under fructose perfusion conditions. The up-regulation as measured by microarray of expression of two genes involved in signal transduction, cyclic nucleotide-gated channel and Blmh, was not confirmed by real-time PCR; PCR also failed to confirm microarray determined up-regulation of cyclic nucleotide-gated channel in previous work (13). However, karyopherin (Kpna2) was confirmed to be up-regulated (P < 0.01) by age and fructose perfusion. Both microarray and real-time PCR analyses also confirmed that β-galactoside 2,6-sialyltransferase 1 (Siat1), a member of the glycosyltransferase family of enzymes, is uniquely up-regulated with age only under fructose perfusion conditions.
All the genes affected by age and identified to be glucocorticoid sensitive by microarray were generally confirmed by real-time PCR. For example, membrane metallo endopeptidase (Mmed) and Pla2g2 were significantly up-regulated by age, and markedly so when the intestine was perfused with fructose. Hsd1b6 was up-regulated, and Sts as well as Srd5a2 were down-regulated by age under both glucose and fructose perfusion. By real-time PCR, Hsd11b2 and Hsd17b2, genes involved in steroid metabolism, appear to be specifically up-regulated by age only under fructose perfusion conditions, confirming microarray results. Glutaminase (Gls) up-regulation was borderline; it was up-regulated by microarray with age under fructose perfusion, but real-time failed to confirm this finding. Instead, real-time PCR found Gls to increase in the intestine of rats perfused with glucose.
A significant number of genes that was specifically responsive to both age and fructose was also identified by Pathway Assist to be regulated by glucocorticoids, suggesting that glucocorticoids may play an important role not only in the development of GLUT5, but also of these genes. Ten of these genes [Mmed, arginase 2 (Arg2), malic enzyme 1 (Me1), Gls, cytochrome P450 2b15 (Cyp2b15), cytochrome P450 subfamily 19 (Cyp19), Myog, Cebp, Hsdd17b2, and Hsd17b6] were up-regulated, and six were down-regulated [Siat1, uncoupling protein 1 (Ucp1), G6pdx, Raf1, syndecan 3 (Sdc3), and syndecan 4 (Sdc4)] by age and fructose perfusion. Because of this interesting link, we reevaluated the role of corticosteroids in intestinal GLUT5 development. We also determined the expression of several genes identified by microarray and Pathway Assist as either being glucocorticoid sensitive or involved in glucorticoid metabolism.
Dex effect parallels the effect of age on GLUT5 development.
Incredibly, dex at doses far below that used for humans allows fructose to enhance GLUT5 expression in 10-d-old pups (compare Fig. 5A with Fig. 2A) at magnitudes similar to those elicited by fructose perfusion of intestines in 20-d-old pups. Without dex, there is no increase in GLUT5 mRNA abundance in the intestine of fructose compared with glucose-perfused pups. In contrast, dex induces a dramatic 35-fold increase in GLUT5 mRNA abundance in fructose-perfused pups and a modest increase of 7-fold (P < 0.01) in glucose-perfused pups. SGLT1 and GLUT2 mRNA abundance each did not vary with dex treatment (P > 0.50) and sugar perfusion (P > 0.10) (Fig. 5, B and C).
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with Fig. 2D). Intestinal glucose uptake increased by 1.6 times (statistically borderline; P = 0.05) in pups perfused with glucose and injected with dex, but there was no effect of dex on glucose uptake in the fructose-perfused pups (Fig. 5E). Proline uptake was independent of dex treatment (P = 0.82) and perfusion solution (P = 0.42) (data not shown), suggesting that dex affects sugar transporters and substantially affects GLUT5 more than SGLT1.
Other genes whose expression are sensitive to both age and dex.
The expression of sucrase isomaltase (Si) increased by 20–30 times with age (Table 5) and was precociously induced by dex 100–300 times. The magnitudes of age and dex effects for Si were similar in both glucose- and fructose-perfused intestines (Fig. 6A). In contrast, the magnitude of age-related increase in mRNA abundance for Arg2 and Mmed expression was greater under fructose perfusion (Table 3). The magnitude of dex-related increase in Arg2 and Mmed mRNA expression was also greater in fructose-perfused intestines (Fig. 6, B and C). Fructose magnified the effect of dex, such that Arg2 expression increased from 15 times in glucose perfused to 45 times in fructose perfused, and Mmed from three times to 10 times. Genes down-regulated with age, like -fetoprotein (Afp) and Hsd11b2 (Table 5), were interestingly not significantly affected by dex (Fig. 6, D and E), although the mRNA abundance of Afp tended to decrease by more than three times with dex and fructose perfusion.
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Fructose-sensitive genes in intestine of 10-d-old rats
Among the fructose-sensitive genes identified by Cui et al. (8) in 20-d-old rat intestine, only cyclin D1 and G6Pc are also regulated by fructose at 10 d old. Interestingly, G6Pc was the only gene up-regulated by fructose after 20-min perfusion in the intestine of 20-d-old pups. The fold increase of G6Pc measured at 10 d old after 4-h fructose perfusion (2.1 times) is similar to the one measured in 20 d old after either 20-min or 4-h fructose perfusion (1.9 times), and much less than that measured at 20 d after 4-h fructose perfusion (10.5 times) (8). The rapid response after just 20-min intestinal exposure to fructose, the magnitude of the response after 4-h perfusion, and the universal response to fructose across various developmental stages indicate that G6Pc has a much higher fructose sensitivity compared with the other genes, including GLUT5. G6Pc is a major regulatory enzyme in the gluconeogenic pathway and may be involved in converting excess fructose to glucose in neonatal enterocytes. Intestinal gluconeogenesis (19) remains a highly controversial issue, with most studies suggesting it does not occur in adults (20, 21) but may occur in neonates (22).
Down-regulated genes may also be important. G6pdx catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconolactone, and in doing so, generates reduced nicotinamide adenine dinucleotide phosphate (NADPH) from nicotinamide adenine dinucleotide phosphate positive (NADP+). The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (Pfkfb4) is responsible for maintaining the cellular levels of fructose-2,6-bisphosphate, which is a key regulator of glycolysis. Therefore, G6pdx and Pfkfb4 are key enzymes regulating intracellular sugar metabolism and contributing to the energy level in the cells. The role of these enzymes and the many others that are regulated differently by fructose and/or glucose would indeed be interesting studies to pursue because of their potential importance to GLUT5 regulation.
Specificity of GLUT5 induction by age and dex
The increasing sensitivity of GLUT5 to fructose with age appears very specific because only GLUT5 and not SGLT1 or GLUT2 gene expression is up-regulated (
50 times) by fructose. Moreover, glucose perfusion does not affect GLUT5 at any age. GLUT2 expression is unchanged between the 10 and 20-d-old rat intestine perfused with either glucose or fructose. Similarly, SGLT1 expression is not affected by age and fructose perfusion. GLUT1 and GLUT8 are age sensitive but not fructose sensitive (supplemental Table 4A). Luminal fructose also increases only fructose uptake at 20 d of age; luminal glucose does not regulate fructose uptake at 10 and 20 d of age. Because glucose uptake is not regulated by fructose, it indicates that the brush border GLUT SGLT1 is not regulated by fructose. Therefore, developmental regulation of GLUT5 is markedly different from SGLT1 and other GLUTs.
Likewise, the role of dex in the fructose-induced increase in GLUT5 expression in 10-d-old pups is also highly specific because SGLT1 and GLUT2 expression is not or only modestly affected by dex, independent of the type of sugar perfused. Dex also allows fructose to only increase fructose and not glucose or proline uptakes (17). Moreover, dex does not allow glucose to increase fructose uptake. Therefore, regulation by glucocorticoids of GLUT5 is specific and markedly different from SGLT1 and other GLUTs.
The role of glucocorticoids in regulating GLUT5 expression during development
Glucocorticoids could be one of the key factors that modulate the ontogenic appearance of GLUT5 in the gut and allow its induction by fructose at 20 but not at 10 d of age. Plasma corticosterone concentration increases dramatically after 14 d of age (23) and may trigger gut maturation. In fact, before weaning in mink and other animals, cortisol generally increased hydrolytic and absorptive capacities of the entire small intestine (24). Therefore, it is important to distinguish the effect of age from the effect of corticosterone during the weaning stage. When gene expression in the small intestine of dex-treated and untreated mice was compared, genes in the development category emerged as being likely candidates for mediation of glucocorticoid-induced maturation of intestinal function (25). Glucocorticoids can modify age-induced changes in meprin β, Gls, and Arg2 expression in neonatal rats (26, 27) and pigs (28), but it is not clear whether their effect is independent of age. In this study dex allowed fructose to enhance GLUT5 expression in 10-d-old pups at magnitudes (20–30 times) similar to those allowed by age in 20-d-old pups, suggesting that dex might mimic the specific effect of age on GLUT5 development.
A previous study by us attempted to distinguish glucocorticoid from age effects by using adrenalectomized rats (29). We showed that in 20-d-old rats adrenalectomized at 10 d old before the endogenous corticosterone surge and exhibiting low corticosterone levels in the plasma, GLUT5 expression and activity were still inducible by fructose. This suggested that fructose used glucocorticoid-independent mechanisms to induce GLUT5 at 20 d of age. However, to avoid salt and water wasting, these adrenalectomized pups received a daily injection of aldosterone. In vivo, mineralocorticoids (aldosterone) and glucocorticoids (corticosterone in rodent) can act in a complementary manner and bind to either the glucocorticoid (GR) or mineralocorticoid (MR) receptors (30). In the study by Monteiro and Ferraris (29), aldosterone may have bound to the MR and/or GR and compensated for the absence of corticosterone, therefore allowing gut maturation and subsequent GLUT5 induction by fructose. Therefore, renewed efforts must be done using adrenalectomized pups to distinguish age from corticosterone effects.
Dex, like corticosterone, is able to bind MR, GR, or the pregnane xenobiotic receptor (31), each of which is expressed in the small intestine of neonatal rats (our unpublished observations). Interestingly, among the age- and fructose-sensitive genes also up-regulated by dex, Arg2 is transcriptionally regulated by glucocorticoids through the intestinal GR (32), whereas CYP3A family members are regulated by pregnane xenobiotic receptor, also in the intestine (33). Moreover, Hsd11b2, which in vivo ensures the selective access of the aldosterone to the MR, is up-regulated by fructose and age. [Because corticosterone and aldosterone bind with equal affinity to MR, the catabolism of corticosterone into inactive 11-dehydro-corticosterone by Hsd11b2 enhances the binding of aldosterone to the MR (30)]. We also identified six potential GR response elements in the GLUT5 promoter regions 0- to 1250-bp upstream from the transcription start site (–128/–119, –306/–297, –551/–542, –1041/–1032, –1238/–1229, and –1246/–1232) using the ElDorado system (www.genomatix.de), suggesting that dex could have a direct nuclear action. However, more studies need to be done to characterize the specific nuclear receptor(s) and response element(s) modulating the effect of dex on GLUT5.
GLUT5 transports a nonessential nutrient that normally appears in the gut lumen only when rats and other omnivorous mammals are weaned, therefore, GLUT5 may require tighter regulation (presence of substrate fructose plus either age or corticosterone) to prevent premature synthesis. More vital transporter genes like SGLT1 and the proline transporter SIT1 are already significantly expressed.
Are Kpna2 and Siat1 involved in GLUT5 development
The failure of fructose to stimulate GLUT5 in intestines of 10-d-old rats could be the result of either inhibitory factors in 10-d-old pups preventing precocious GLUT5 stimulation by fructose, or stimulatory factors appearing only at 20 d of age, and allowing GLUT5 transcription. Two genes, each confirmed by microarray and real-time PCR to be simultaneously fructose responsive and age dependent, may be one of these factors. Kpna2 is overexpressed with age and could be involved in GLUT5 stimulation in 20 d old, whereas Siat1, highly expressed in 10-d-old rat intestines perfused with fructose, could act as an inhibitor of GLUT5 regulation at this age.
Kpna2 is a nuclear import receptor belonging to the importin -family and implicated in the transport through the nuclear envelope of more than 45 kDa proteins (34). GRs enter the nucleus along with importin proteins. Kpna2 may be linked to GLUT5 regulation because its mRNA is overexpressed in normal testis and malignant breast tumors (35), tissue types characterized by high levels of GLUT5 mRNA or protein (36, 37). Moreover, metabolizable sugars induced Kpna2 translocation from the hepatocyte nucleus to the cytoplasm (38, 39). This glucose- and fructose-induced movement of Kpna2 is energy dependent but wortmannin insensitive (38). In neonatal rats, fructose-induced GLUT5 expression is also wortmannin insensitive (8). Kpna2 proteins are also known to be tethered to GLUT2 in hepatoma cells, which do not express GLUT5, and may be involved in transmission of the signals that regulate expression of glucose-sensitive genes (39).
Siat1 transfers sialic acid groups to cell-surface glycoproteins and glycolipids, thereby affecting their function (40). The progressive loss of sialic acids in brush-border membrane glycoproteins is one of the major changes occurring in the rat (Table 6) and mouse (41) small intestine during the transition from suckling to weaning. Reduced sialylation with intestinal maturation is the result of a lower expression of Siat1 in weaning rats. Precocious reduction of Siat1 levels could be induced earlier by glucocorticoids (41, 42). Interestingly, sialylation could also affect nuclear proteins, and play a role in signal transduction and in repression or activation of gene expression (43). In fact, inhibiting glycosylation of the transcription factor SP1 decreases the expression of membrane transport proteins. Therefore, one of the hypotheses to explain the regulation of GLUT5 during development could be that the decrease in Siat1 expression with age leads to a loss of sialic acids on the nuclear factor(s) involved in GLUT5 transcription.
Caveats, relevance, and future studies
Although we found remarkable differences in expression of intestinal genes between 10- and 20-d-old pups, and between 10-d-old pups perfused with fructose and glucose, the microarray was based on a 5K chip, and, therefore, the list is incomplete. However, the primary objective of the study was not to compile a complete list of age- or fructose-responsive genes (already there are a large number) but to identify pathways involved in GLUT5 development, which can be accomplished by analyzing interactions among a smaller number of genes. For example, the important role of glucocorticoids was discovered when a significant number of fructose- and age-sensitive genes was identified to also be sensitive to glucocorticoids by computer tools that visualize biological pathways and gene regulation networks. The second concern is that the RNA was sampled from a heterogenous cell population. Cell culture approaches do provide a homogenous cell population but cannot be used to study ontogenetic development. In this in vivo study, only the intestinal epithelial layer consisting of more than 80% enterocytes was exposed to differences in luminal sugar; whereas most other cell types were not. Moreover, only the intestinal mucosa was used in this study, thereby eliminating muscle and connective tissue layers, so differences in expression as we have already shown by in situ hybridization (6) and immunocytochemistry (8) would have occurred from the differences in response by enterocytes. In future work, colocalization of age-, dex- or fructose-responsive genes with GLUT5 will confirm their expression in enterocytes. Finally, intestinal development may involve activation (e.g. phosphorylation) of some proteins (44), and not necessarily changes in mRNA expression, therefore, microarray analysis would be unable to detect those changes. However, genes downstream of these activated proteins may still change in expression and would be detected by microarray.
Although they account for less than 10% of births, preterm infants in the United States account for half of infant hospitalization costs occurring, mainly from respiratory distress and necrotizing enterocolitis (45). Because prenatal corticosteroid treatment stimulates lung surfactant synthesis in the preterm infant, prenatal mothers at risk for premature delivery and/or preterm infants receive corticosteroids (46), which as we have clearly shown in this study, also affect the maturation of the fructose transporter system in the small intestine. Biologically, the intestine’s capability to reprogram indicates developmental plasticity that allows a range of phenotypes to develop from a single genotype (47). Plasticity provides organisms with the ability to change function in response to environmental cues. When the dam is under stress, it may release high levels of glucocorticoids in the milk (48) that subsequently and precociously enable the small intestine of the pup to digest and absorb nutrients obtained from the environment, thereby enhancing its survival in case the dam becomes malnourished or dies.
In conclusion, microarray in this study permitted us to identify a pathway, involving glucocorticoids, that participates in the ontogenetic development of GLUT5. In vivo, the timing of the increases in glucocorticoids and GLUT5 fructose sensitivity suggests that GLUT5 gene expression is glucocorticoid dependent at the early stage of neonatal rats. For the first time, the role played by glucocorticoids in GLUT5 regulation in the small intestine has been clearly demonstrated. The potential interaction of signaling factors such as Kpna2 or Siat1 with the glucocorticoids needs to be explored, as well as determining the nuclear receptor involved in glucocorticoid induction of GLUT5.
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This work was supported by NIH Grant RDK075617A and NSF Grant IBN-722365 (to R.P.F.) and by the Philippe Foundation (to V.D.).
Disclosure Statement: The authors have nothing to declare.
First Published Online October 18, 2007
Abbreviations: Arg2, Arginase 2; Afp, -fetoprotein; Blmh, bleomycin hydrolase; Cebpd, CCAAT/enhancer binding protein (C/EBP), 
; Cyp2b15, cytochrome P450, 2b15; Cyp19, cytochrome P450, subfamily 19; dex, dexamethasone; EF1, elongation factor 1; G6Pc, glucose-6-phosphate, catalytic; G6pdx, glucose-6-phosphate dehydrogenase; Gls, glutaminase; GLUT, glucose transporter; GLUT2 (Slc2A2), basolateral glucose/fructose transporter; GLUT5 (Slc2A5), intestinal fructose transporter; GLUT7 (Slc2A7), facilitative hexose transporter; GO, gene ontology; GR, glucocorticoid receptor; Hsd3b1, 3β-hydroxysteroid dehydrogenase 1; Hsd11b2, hydroxysteroid 11-β dehydrogenase type 2; Hsd17b2, 17-β hydroxysteroid dehydrogenase type 2; Hsd17b6, 17-β hydroxysteroid dehydrogenase type 6; 10F or 10G, intestine of 10-d-old pups perfused with fructose or glucose, respectively; 20F or 20G, intestine of 20-d-old pups perfused with fructose or glucose, respectively; Kpna2, karyopherin; Me1, malic enzyme 1; Mmed, membrane metallo endopeptidase; Myog, myogenin; MR, mineralocorticoid receptor; Nr1h4, nuclear receptor subfamily 1, group H, member 4; Nucb, nucleobinding; Pfkfb4, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4; Pla2g2a, phospholipase A2, group IIA; Rdh2, retinol dehydrogenase type 2; Sdc3, syndecan 3; Sdc4, syndecan 4; Si, sucrase isomaltase; Siat1, β-galactoside 2,6-sialyltransferase 1; SGLT1, sodium-glucose cotransporter 1; Srd5a2, steroid 5--reductase 2; UCP1, uncoupling protein 1.
Received July 5, 2007.
Accepted for publication October 11, 2007.
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and β subunits. FEBS Lett 462:368–372[CrossRef][Medline]2 nuclear transport pathway is regulated by glucose in hepatic and pancreatic cells. Traffic 5:10–19[CrossRef][Medline]2: a control step of glucose-sensitive gene expression in hepatic cells. Biochem J 364:201–209[CrossRef][Medline]-2,6-sialyltransferase during normal and cortisone-induced maturation in mouse intestine. Am J Physiol Gastrointest Liver Physiol 282:G480–G4902,6-sialyltransferase in the small intestine of suckling rats is suppressed by mifepristone (RU-38.486). J Steroid Biochem Mol Biol 60:59–66[CrossRef][Medline]This article has been cited by other articles:
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  V. Douard and R. P. Ferraris Regulation of the fructose transporter GLUT5 in health and disease Am J Physiol Endocrinol Metab, August 1, 2008; 295(2): E227 - E237. [Abstract] [Full Text] [PDF]
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 V. Douard, H.-I. Choi, S. Elshenawy, D. Lagunoff, and R. P. Ferraris Developmental reprogramming of rat GLUT5 requires glucocorticoid receptor translocation to the nucleus J. Physiol., August 1, 2008; 586(15): 3657 - 3673. [Abstract] [Full Text] [PDF]
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