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Am J Physiol Gastrointest Liver Physiol 288: G1310-G1320, 2005. First published February 3, 2005; doi:10.1152/ajpgi.00550.2004
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HORMONES AND SIGNALING

Fructose-induced increases in neonatal rat intestinal fructose transport involve the PI3-kinase/Akt signaling pathway

Xue-Lin Cui, Anna M. Schlesier, Elda L. Fisher, Carla Cerqueira, and Ronaldo P. Ferraris

Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey

Submitted 14 December 2004 ; accepted in final form 28 January 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Expression of rat glucose transporter-5 (GLUT5) is tightly regulated during development. Expression and activity are low throughout the suckling and weaning stages, but perfusion of the small intestinal lumen with fructose solutions during weaning precociously enhances GLUT5 activity and expression. Little is known, however, about the signal transduction pathways involved in the substrate-induced precocious GLUT5 development. We found that wortmannin and LY-294002, inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) specifically inhibited the increase in fructose uptake rate and brush-border GLUT5 protein abundance but not GLUT5 mRNA abundance. Perfusion of EGF, an activator of PI3-kinase, also resulted in a marked wortmannin-inhibitable increase in fructose uptake. Perfusion of fructose for 4 h increased cytosolic immunostaining of phosphatidylinositol-3,4,5-triphosphate (PIP3), the primary product of PI3-kinase, mainly in the mid- to upper-villus regions in which the brush-border membrane also stained strongly with GLUT5. Perfusion of glucose for 4 h had little effect on fructose or glucose uptake and PIP3 or GLUT5 staining. SH-5, an Akt inhibitor, prevented the increase in fructose uptake and GLUT5 protein induced by fructose solutions, and had no effect on glucose uptake. The PI3-kinase/Akt signaling pathway may be involved in the synthesis and/or recruitment to the brush border of GLUT5 transporters by luminal fructose in the small intestine of weaning rats. Increases in fructose transport during the critical weaning period when rats are shifting to a new diet may be modulated by several signaling pathways whose cross talk during development still needs to be elucidated.

development; glucose; intestine; epidermal growth factor; mucosa

BECAUSE OF DRAMATIC INCREASES in consumption of soft drinks and fruit juices, per capita consumption of fructose in the United States has increased by 10 times in 30 yr to almost 60 g fructose per day (4, 39). Paralleling this remarkable increase in fructose consumption is an alarming increase in incidence of obesity and in prevalence of type II diabetes (4, 14). What makes this correlation disturbing is that per capita fructose consumption in very young children has increased faster than that of the general population and in the 1980s was already ~30–40 g fructose per day representing 10% of their energy intake (39). The top 10th percentile of subjects in all age groups typically consume approximately two times more fructose, exposing this particular age group (1–6 yr of age) to potential metabolic derangements caused by excessive fructose consumption. There are very few studies on physiological adaptations to excessive fructose consumption, and even fewer on intestinal fructose absorption, particularly in neonates.

The classical model of intestinal sugar transport depicts glucose and galactose to be transported by the sodium-dependent glucose transporter 1 (SGLT1) from the lumen into the cytosol, whereas fructose is transported by a facilitated, sodium-independent glucose transporter (GLUT5) (16, 54). Glucose, galactose, and fructose are subsequently released from the cytosol into the blood by a different facilitative glucose transporter (GLUT2) located in the basolateral membrane. By way of these transporters, transcellular active transport becomes the predominant mechanism of in vivo glucose absorption under physiological conditions (22). Unlike the other sugar transporters SGLT1 and GLUT2, GLUT5 activity and expression are tightly regulated by development, that is, they are underexpressed in the neonatal rat small intestine until weaning is completed (48) but that expression can be precociously enhanced during weaning if fructose is provided (27). Previous studies have demonstrated that the GLUT5 activity and mRNA expression are regulated by luminal fructose itself (5, 44). However, the signal transduction mechanisms involved in the fructose-induced upregulation of GLUT5 activity and expression in the small intestine of the neonatal rats are still poorly investigated.

The phosphatidylinositol 3-kinase/protein kinase B (PI3-kinase/Akt) signal transduction pathway plays an important role in glucose metabolism and is involved in regulating glucose uptake in insulin-responsive tissues, in activating glycogen synthesis, and in inducing glycolysis (12, 29). Fructose-feeding increases the activity of PI3-kinase (3) and has recently been shown to alter the phosphorylation of the regulatory subunit of PI3-kinase (45) in rat skeletal muscle. Therefore, we hypothesized that the PI3-kinase/Akt signaling pathway plays a major role in the fructose-induced upregulation of GLUT5 activity. The role of the PI3-kinase/Akt pathway in regulating GLUT5 activity and gene expression during development has not yet been investigated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental design. Wortmannin and LY-294002 (Biomol Research Laboratories, Plymouth Meeting, PA) inhibit, whereas EGF activates PI3-kinase. SH-5 is a specific inhibitor of Akt. Twenty-day-old weaning rat pups were divided into four perfusion groups: G (glucose), G + I (glucose + inhibitor), F (fructose), and F + I (fructose + inhibitor). In the first 0.5 h, G and F intestines were initially perfused with vehicle in Krebs-Ringer bicarbonate (KRB), whereas G + I and F + I intestines were perfused with inhibitors in KRB (KRB + I) solutions (Fig. 1). Both SH-5 and EGF were soluble in aqueous solutions. Wortmannin and LY-294002 were initially dissolved in DMSO but such low concentrations were used that microliter amounts were added to 4 liters of perfusion solution; "vehicle" solutions included DMSO without the inhibitors. The solutions were then changed to glucose (G), glucose + inhibitor (G + I), fructose (F), and fructose + inhibitor (F + I) in KRB solutions that were continuously perfused for 4 h. In the EGF experiment, rats were separated into four groups: KRB, KRB + Wortmannin, EGF, and EGF + wortmannin and the intestines of the rats were perfused for 4 h. After the experiments, samples were collected for measuring sugar uptake rates, for storing in liquid nitrogen for protein analysis, and for storing in a –80°C freezer for subsequent RNA analysis.



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  		Fig. 1. Experimental design. To completely block the PI3-kinase/Akt signaling transduction pathway in the intestinal epithelial cells, the inhibitors of PI3-kinase and Akt were first perfused for 0.5 h through the lumen, and then the solutions of sugar or EGF plus inhibitors were subsequently perfused for 4 h. KRB, Krebs-Ringer bicarbonate solution; G, glucose; F, fructose; I, inhibitor; W, wortmannin. n = 5–6 Rats in each group.

 
The perfusion duration and sugar concentration were based on previous studies (19, 27, 43). The concentration of sugar was based on findings that the magnitude and time course of enhancement of GLUT5 mRNA abundance and fructose transport rates with 100 mM fructose perfusion were the same as those observed when pups were fed 65% (wt/wt) fructose pellets (27). Intestinal luminal sugar concentration in pups fed pellets containing 65% (wt/wt) sugars can exceed 100 mM (19). A 100 mM glucose or fructose solution was perfused to yield the maximum effects of the sugars on sugar transport and transporter gene expression. Wortmannin (IC50 = 1 µM) was perfused at a concentration of 10 nM because wortmannin is effective from 10 to 100 nM in blocking the activity of PI3-kinase in intestinal tissues (31). LY-294002 (IC50 = 1 µM, Biomol Research Laboratories), can inhibit intestinal PI3-kinase at 1–25 µM (1). However, preliminary work showed that a 4-h in vivo perfusion of >0.1 µM LY-294002 increased mortality rate during perfusion. We therefore chose 0.1 µM not only because this low concentration minimized mortality rate during perfusion but also because it clearly decreased the fructose-induced increase in fructose transport.

To determine the role of Akt (or protein kinase B), we used its specific, cell-permeable inhibitor, SH-5. Because this inhibitor has not yet been used in the intestine, a series of bioassay experiments (0.02, 0.2, and 2 µM) were also performed to determine the optimal concentration. We found that 0.2 µM was the concentration most effective in blocking the fructose-induced increase in fructose uptake in the neonatal rat small intestine. These concentrations had no effect on mortality rate during perfusion.

In the EGF experiments, the small intestine was perfused with 60 ng/ml of EGF, a concentration effective in modulating intestinal glucose transport in adult rats (36).

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 (UMDNJ-NJMS). Adult male and female Sprague-Dawley rats weighing ~200 g were purchased from Taconic (Germantown, NY) and bred in the Research Animal Facility of UMDNJ-NJMS. 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. In this study, 20- to 22-day-old midweaning rat pups were used. They were randomly separated into four groups according to experimental designs described in Fig. 1. Before the experiments, all rats were housed in a temperature-controlled room (22–24°C) with a 12:12-h light-dark cycle, and allowed access to water and nonpurified diet ad libitum (Purina Mills, Richmond, IN).

Perfusion model. Rat intestinal perfusion procedure was conducted following the method of Jiang and Ferraris (27). Briefly, rat pups (20- to 22-day-old, not starved) were anesthetized by intraperitoneal injection of urethane in 0.9% saline (1.4 g/kg body wt). The abdominal cavity was opened by making a midline abdominal incision, and the small intestine with intact blood vessels and nerve connections was exposed. A small incision was made at ~5 cm distal to the ligament of Treitz, and a catheter was inserted into the lumen and then secured with surgical thread. A plastic tube (Tygon; 0.8-mm ID) was catheterized into the ileum 10 cm proximal from the ileocecal valve. After the contents were flushed with regular KRB solution (in mM: 128 NaCl, 4.7 KCl, 2.5 CaCl2·5H2O, 1.2 MgSO4, 19 NaHCO3, 2.2 KH2CO3), the small intestine was continuously perfused with sugar solution (100 mM sugar in KRB) 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 KH2CO3, and 100 glucose or fructose. Rat pups were kept under continuous anesthesia by adding 25% (wt/vol) urethane in 0.9% saline into the abdominal cavity every 30 min. Body and perfusion solution temperatures were maintained at 37°C by heating pads and water baths, respectively. After the experiments, sugar uptakes, transporter protein, and mRNA abundance were measured.

Fructose and glucose uptake measurements. Measurements of glucose and fructose uptake rates were performed by following the technique of Karasov and Diamond (28). Briefly, after perfusion, the jejunum was immediately isolated and gently flushed with ice-cold (2°C) KRB. Four 1-cm segments were everted and mounted on a grooved steel rod (3 mm diameter) and preincubated at 37°C for 5 min in KRB solution bubbled with 95% O2-5% CO2 (vol/vol). The sleeves were then incubated at 37°C in an oxygenated, stirred (1,200 rpm) solution containing either [D-14C]-labeled glucose for 1 min or [D-14C]-labeled fructose for 2 min. [L-3H]-labeled glucose was used to correct for adherent fluid and passive diffusion of glucose or fructose. To reduce the radioactive label in the adherent fluid, there was a 20-s rinse in 30 ml ice-cold KRB solution immediately after the incubation. All radioisotopes were purchased from New England Nuclear (Boston, MA). Results were expressed as nanomoles per milligram wet weight of intestine. For each rat, two sleeves were used for measuring uptake rates of each test solute. The average was calculated to represent the glucose or fructose absorption in that rat. Fructose and glucose uptake rates were determined at 50 mM, which yields near maximum velocity (Vmax) rates, so that any change in uptake rate will be due mainly to a change in Vmax (28).

Total RNA extraction. At the end of each perfusion, ~10 cm of jejunum 15 cm from the ligament of Treitz was quickly frozen in liquid nitrogen and then stored at –80°C for later RNA extraction. Total RNA was extracted using a commercially available reagent TRIzol (GIBCO-BRL, Gaithersburg, MD). Briefly, 50–100 mg of tissue was homogenized in 1 ml of TRIzol reagent and processed according to the kit manufacturer's instructions. After the concentration of the total RNA was measured by spectrophotometry (Beckman Instruments, Fullerton, CA), the sample was stored in –80°C freezer for the subsequent Northern blot analysis.

Northern blot analysis. Northern blots and semiquantitative PCR were used to quantify mRNA abundance, and results from one method typically confirmed that of the other. For Northern blots, total RNA (30 µg) from each sample were subjected to 1% agarose-6% formaldehyde electrophoresis and then transferred to a nitrocellulose membrane by capillary action. After the RNA was UV cross-linked on the membrane and dried for 2 h, cDNA probes of rat GLUT5 and 18S were labeled with [32P]dCTP using a random primer labeling kit (RTS RadPrime DNA labeling system; GIBCO-BRL). Hybridization and quantification followed methods previously described (7, 27) The ratio of GLUT5 mRNA to 18S rRNA (loading and transfer control) abundance was calculated to represent relative GLUT5 gene expression in the intestine.

RT-PCR. Semiquantitative RT-PCR was employed to quantify the mRNA expression of a target gene using a OneStep RT-PCR kit (Qiagen). Briefly, the reaction was performed on a PCR thermal cycler (Techne; Duxford Cambridge, UK) for 30 min at 50°C, 15 min at 95°C for RT reaction and activation of Taq DNA polymerase. The DNA amplification procedure was set at 45 s, 94°C (denature); 45 s, 60–65°C (annealing); and 90 s, 72°C (extension) for a total number of cycles optimal for PCR amplification of different genes. The reaction mixture was stocked at 4°C after a final polymerization of 10 min at 72°C. The PCR product was loaded onto 1% agarose gel for electrophoresis and stained with ethidium bromide. The gel pictures were taken by a charge-coupled device (CCD) camera for determination of the density of the bands by a densitometer (FluorChem 8800; Alpha Innotech).

After the optimal number of PCR cycles for each gene was determined in pilot experiments (9), RT-PCR was carried out. The relative amount of mRNA in the samples for each target gene measured was normalized by a housekeeping gene GAPDH and presented as a ratio to GAPDH (20, 37, 51). Primers used for detecting the mRNA expression of PI3-kinase p85{alpha}, p110{alpha}, and GAPDH in this study were as follows: PI3-kinase p85{alpha} (GenBank accession no. D64045): forward: 5'-TCACCGAGATGGGAAATACG-3', reverse: 5'-TACAGAGCAGGCATAGCAGC-3'(PCR product: 832 bp); PI3-kinase p110{alpha} (GenBank accession no. AF395897): forward: 5'-TCAAGCAGGAGAAGAAGGATG-3', reverse: 5'-GTCAAAACAAACGGCACACG-3' (715 bp); and GAPDH (GenBank accession no. NM_017008): forward: 5'-TGAAGGTCGGTGTCAACGGATTTGGC-3', reverse: 5'-CATGTAGGCCATGAGGTCCACCAC-3' (983 bp).

Preparation of brush-border membrane vesicles. After perfusion, the intestinal mucosa was immediately scraped from 10 cm of jejunum, snap frozen in liquid nitrogen, and then stored at –80°C. The mucosa was subsequently placed in 20 ml of ice-cold mannitol/Tris buffer (300 mM mannitol, 1 mM Tris·HCl, pH 7.4) and homogenized with a Polytron homogenizer (setting 9) for 30 s. Before adding CaCl2/Tris solution (100 mM CaCl2, 1 mM Tris, pH 7.4) to make a final concentration of Ca2+ < 10 mM, 1 ml of the homogenate was collected for later protein quantification and alkaline phosphatase activity assay. The solution was stirred on ice for 15 min and then centrifuged at 2,000 g (model RC-5; Kendro Laboratory Products, Newtown, CT) at 2°C for 15 min to remove nuclei and large cellular debris. The supernatant was further centrifuged twice at 37,000 g at 2°C for 30 min, and the pellet was washed in 2 ml mannitol/Tris buffer. The vesicle preparation was then suspended in 1 ml of mannitol/Tris buffer with an ultrasonicator (Branson Ultrasonics, Danbury, CT). The protein concentration in the brush-border membrane vesicle (BBMV) and homogenate samples was measured by a DC Protein Assay method following the manufacturer's instruction (Bio-Rad Laboratories, Hercules, CA).

Activity of alkaline phosphatase was determined in the homogenate and BBMV samples following earlier work in the laboratory (18). The specific activity of alkaline phosphatase was typically at least ~10-fold higher in the BBMV compared with that in the homogenate sample. These data indicated that the prepared BBMV sample was indeed enriched in apical membrane vesicles.

Western blot analysis. Total protein (40 µg) from BBMV and homogenate samples were mixed with 2x Laemmli sample buffer (Sigma-Aldrich, St. Louis, MO) and run on a 10% SDS-PAGE using a Mini-PROTEAN II cell (Bio-Rad Laboratories) for 2 h. The proteins were transferred onto a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ) for 1 h at room temperature using the model 6000 Electroblotter (E & K Scientific Products, Saratoga, CA). Once the membrane with blots was prepared, the following steps were conducted. First, the membrane was blocked in 5% nonfat milk in Tris-buffered saline-Tween 20 (TBS-T) (0.1% Tween 20, 50 mM Tris, 137 mM NaCl, pH 7.4) buffer for 1 h. Second, the blots were incubated with primary antibody (rabbit anti-rat GLUT5; catalog no. AB1348; epitope is the COOH-terminal region of rat GLUT5), rabbit anti-rat GLUT2 (catalog no. AB1342, epitope is the exoplasmic loop between helices 1 and 2 of rat liver GLUT2) or {beta}-tubulin polyclonal antibody (Chemicon International, Temecula, CA) diluted 1:1,000 in TBS-T buffer for 1 h. Third, the membrane was incubated in secondary antibody solution, in which the goat anti-rabbit IgG coupled to horseradish peroxidase was diluted as 1:2,000 in TBS-T, for 30 min. The membrane was washed (5 min) three times in TBS-T between the treatments. Finally, after the membrane was treated with the enhanced chemiluminescence detection solution (Amersham Biosciences), it was exposed to the Kodak BioMax film (Amersham Biosciences) for 0.2–5 min. The film was developed, and the immunoblots were scanned with a CCD camera. The density of the blots was quantified by using a densitometry system (model IS-1,000 Digital Imaging System, Alpha Innotech). Results were reported as percentage of protein abundance in other samples relative to that in the glucose-perfused sample.

Immunohistochemistry. Two centimeters of jejunum segment ~10 cm from the Treitz's ligament of each rat were excised and placed immediately into ice-cold 4% buffered formalin solution overnight. After the tissue was washed four times in 70% ethanol, it was dehydrated and then embedded in paraffin (Technicon, Tarrytown, NY). Five micrometer sections were cut and adhered on slides by baking at 65°C for 1 h. A commercially available immunostaining kit (Vector Laboratories, Burlingame, CA) was employed for GLUT5 and PIP3 detection in the intestinal tissue following earlier work (46). For negative control in the immunostaining procedures performed, rabbit normal serum (or antibody plus peptide used to raise the antibody) replaced the primary antibodies.

Statistical analysis. Data are presented as means ± SE. A one- or two-way ANOVA was first used to determine the significance of the difference of relative mRNA abundance among groups with different treatments. If there was a significant difference, Fisher's paired least significant difference test was used to determine the particular effect that caused that difference. Unpaired Student's t-test was used to determine the difference between fructose-perfused and glucose-perfused groups, or drug treated and untreated groups. P < 0.05 was considered statistically significant. Statistical analysis used the StatView program (Abacus Concepts, Berkeley, CA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of wortmannin on fructose-induced fructose transport. GLUT5 mRNA expression was significantly greater (>2.2-fold, P < 0.001) in the neonatal rat small intestine perfused with fructose compared with that perfused with glucose (Fig. 2). However, there was no significant difference for both SGLT1 (P = 0.65) and GLUT2 (P = 0.88) mRNA expression between glucose- and fructose-perfused intestines. The fructose-induced increase in GLUT5 mRNA abundance was not blocked by wortmannin (P = 0.57). Wortmannin did not affect the expression of SGLT1 (P = 0.73) and GLUT2 (P = 0.40) mRNA expression.



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  		Fig. 2. Fructose (100 mM; 4-h perfusion) markedly increased fructose transporter GLUT5 but not sodium-dependent glucose transporter SGLT1 and facilitated glucose transporter GLUT2 mRNA abundance in the small intestine of neonatal rats. Wortmannin (10 nM) did not affect the fructose-induced increase in GLUT5 mRNA abundance. A: representative Northern blot. B: relative GLUT5, SGLT1, and GLUT2 mRNA abundance. Bars are means ± SE (n = 5–6). The ratio of sugar transporter mRNA abundance in glucose-perfused (control) intestine was designated as 100%, and the ratios in other groups were normalized to this value. Different letters indicate a significant difference (P < 0.05) in mRNA abundance.

 
GLUT5 protein also increased ~2.2-fold (P < 0.01) in the homogenate of the fructose-perfused small intestinal mucosa compared with the glucose-perfused small intestinal mucosa, consistent with the twofold increase of GLUT5 mRNA abundance induced by the fructose perfusion (Fig. 3). In addition, GLUT5 protein also increased ~1.5-fold (P < 0.05) in BBMVs of fructose-perfused small intestine. The fructose-induced increase in GLUT5 protein abundance was inhibited by wortmannin in both homogenate (P < 0.01) and BBMV (P < 0.05) preparations. {beta}-tubulin was abundant in the homogenate but not in BBMV, indicating that contamination of BBMV samples with cytosolic proteins was minimal.



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  		Fig. 3. Wortmannin (10 nM) blocked the fructose-induced increase in GLUT5 protein in both homogenates and brush-border membrane vesicles (BBMVs) prepared from the small intestinal mucosa of neonatal rats. A: representative Western blot. B: relative density of GLUT5 protein. Bars are means ± SE (n = 3). The density of GLUT5 protein in glucose-perfused (control) intestine was designated as 100%, and the densities in other groups were normalized to this value. Different letters indicate a significant difference in transporter abundance.

 
The GLUT5 protein was expressed in extremely low amounts in the homogenates of liver and primary hepatocytes (Fig. 4A). Similar low amounts were also in intestinal homogenates of glucose-perfused rats. Consistent with the above experiment (Fig. 3), GLUT5 protein was much higher in the homogenates of fructose-perfused compared with that in the glucose-perfused intestine. The abundance of GLUT5 protein was even much greater in intestinal BBMVs than in homogenates.



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  		Fig. 4. Western blots depicting sugar transporters in the liver and intestine of neonatal rats. Lanes 1 and 2: liver homogenates. Lanes 3 and 4: homogenates from primary hepatocytes. Lane 5: homogenate from glucose-perfused intestinal mucosa. Lane 6: BBMV from glucose-perfused intestinal mucosa. Lane 7: homogenate from fructose-perfused intestinal mucosa. Lane 8: BBMV from fructose-perfused intestinal mucosa. GLUT5, 55 kDa; GLUT2, 61 kDa; SGLT1, 75 kDa and {beta}-tubulin, 51 kDa. A: exposure for 8 min; B: exposure for 1 min.

 
As would be expected, GLUT2 protein was expressed in liver tissue and in primary hepatocytes (positive controls) (Fig. 4A). GLUT2 was also clearly found in apparently similar quantities in homogenates of both glucose- and fructose-perfused intestines. In contrast, GLUT2 was in vanishingly low abundance in BBMVs prepared from the mucosa of the same intestinal samples. GLUT5 and GLUT2 protein abundance in homogenates of glucose-perfused intestines was similarly very low; however, GLUT5 protein abundance was much greater than that of GLUT2 protein in homogenates of fructose-perfused intestines, suggesting that GLUT5 but not GLUT2 specifically responds to luminal fructose when glucose-perfused intestine is used as control. Compared with GLUT5 and GLUT2 in homogenates, and GLUT2 in BBMVs, GLUT5 was dramatically greater in intestinal BBMVs, to the point that the X-ray film was saturated and it was impossible to determine differences in GLUT5 abundance in BBMVs (Fig. 4A). When exposure duration was reduced eightfold (Fig. 4B), there was a difference in BBMV GLUT5 abundance between homogenates and BBMVs of fructose- and glucose-perfused intestines. These results indicate that GLUT2 protein in these in vivo preparations was located mainly in the basolateral and not in the brush-border membrane and that GLUT5 is the predominant fructose transporter located in the brush-border membrane of neonatal small intestinal absorptive cells.

The SGLT1 protein was detectable in the BBMV of small intestinal epithelial cells from neonatal rats, but there was no significant difference between glucose- and fructose-perfused intestines (Fig. 4). As would be expected, SGLT1 was not localized in the homogenate of small intestinal mucosa, liver tissue, and primary hepatocytes. Cytosolic protein {beta}-tubulin was found in homogenates of liver, primary hepatocytes, and small intestinal mucosa. However, it was undetectable in the BBMV samples extracted from the small intestinal mucosa.

As with our previous studies, fructose perfusion significantly increased fructose uptake rate (P < 0.01), but it did not influence the glucose uptake rate (P = 0.40) (Fig. 5). The increase (~1.7-fold) of fructose uptake induced by luminal fructose was consistent with the increase (~2-fold) of GLUT5 protein in the BBMV induced by the same solution. Wortmannin blocked the fructose-induced increase in fructose uptake rate (P < 0.01 compared with uptake in high fructose), but had no effect on glucose uptake rate. To confirm the effect of wortmannin on fructose uptake, we used another specific inhibitor of PI3-kinase, LY-294002, and found similar results (Fig. 6).



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  		Fig. 5. Wortmannin blocked the fructose-induced increase in fructose uptake but did not affect glucose uptake in the small intestine of neonatal rats. Bars are means ± SE (n = 6). Uptake rate in glucose-perfused (control) rats was designated as 100%, and the uptake rates in other rats were normalized to this value. Different letters indicate a significant difference in uptake rates.

 


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  		Fig. 6. LY-294002 (0.1 µM), another inhibitor of PI3-kinase, blocked the fructose-induced increase in fructose uptake but did not affect glucose uptake in the small intestine of neonatal rats. Bars are means ± SE (n = 7). Uptake rate in glucose-perfused (control) rats was designated as 100%, and the uptake rates in other rats were normalized to this value. Different letters indicate a significant difference in uptake rates.

 
Effect of fructose on PI3-kinase p110{alpha} and p85{alpha} mRNA expression and on PIP3 abundance. Although GLUT5 mRNA abundance increased approximately fourfold (P < 0.001) in the small intestine, fructose perfusion had no effect on the mRNA abundance of PI3-kinase subunits p110{alpha} (P = 0.34) and p85{alpha} (P = 0.11) (Fig. 7). We also quantified the mRNA abundance of PI3-kinase p110{alpha} and p85{alpha} with Northern blot analysis by using probes generated from the above RT-PCR and obtained the same results (data not shown).



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  		Fig. 7. Fructose increased GLUT5, but not PI3-kinase p110{alpha} and p85{alpha}, mRNA in the small intestine of neonatal rats as determined by semiquantitative RT-PCR. A: gel pictures. M, DNA size marker. B: mean relative abundance ± SE (n = 5). The expression of GLUT5, phosphatidylinositol 3-kinase (PI3-kinase) p110{alpha} and p85{alpha} (represented as the ratio of each gene to GAPDH) in glucose-perfused pups was designated as 100%, and then its expression in fructose-perfused pups was normalized to this value. Different letters indicate a significant difference in mRNA abundance.

 
GLUT5 protein was in the brush-border membrane of the small intestinal epithelial cells (Fig. 8). In contrast, PIP3, the product of PI3-kinase, was only detected in the cytosol. Both the GLUT5 protein and PIP3 were strongly stained in the fructose-perfused intestine when compared with the glucose-perfused intestine. Hence, there is a correlation between GLUT5 and PIP3 staining in the small epithelial cells perfused with fructose solutions.



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  		Fig. 8. Fructose perfusion increased GLUT5 protein (brown staining in brush border) and phosphatidylinositol-3,4,5-triphosphate (PIP3) (blue staining in cytosol) abundance in the small intestinal epithelial cells. Bars, 150 µm; inset: 20 µm.

 
Effect of SH-5 on fructose-induced fructose transport. GLUT5 (P < 0.001) but not SGLT1 (P = 0.09) and GLUT2 (P = 0.15) mRNA expression increased significantly (>2-fold) in the neonatal rat small intestine perfused with fructose (Fig. 9). The fructose-induced increase in GLUT5 mRNA abundance was not blocked by SH-5 (P = 0.51). Furthermore, SH-5 did not affect the expression of SGLT1 (P = 0.97) and GLUT2 (P = 0.85) mRNA expression.



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  		Fig. 9. Fructose markedly increased GLUT5, but not SGLT1 and GLUT2, mRNA abundance in the small intestine of neonatal rats. SH-5 (10 nM) did not affect the fructose-induced increase in GLUT5 mRNA abundance. A: representative Northern blot. B: relative GLUT5, SGLT1, and GLUT2 mRNA abundance. Bars are means ± SE (n = 5–6). The ratio of sugar transporter mRNA abundance in glucose-perfused (control) intestine was designated as 100%, and the ratios in other groups were normalized to this value. SH, SH-5, an inhibitor of Akt. Different letters indicate a significant difference in mRNA abundance.

 
GLUT5 protein increased ~3.4-fold (P < 0.01) in the homogenate and ~2.3-fold (P < 0.01) in the BBMVs of fructose-perfused intestinal mucosa. This fructose-induced increase in GLUT5 protein abundance was completely blocked by SH-5 in both homogenate (P < 0.01) and BBMV (P < 0.01) fractions (Fig. 10).



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  		Fig. 10. SH-5 (10 nM), an inhibitor of Akt, blocked the fructose-induced increase in GLUT5 protein in both homogenate and BBMV prepared from the small intestinal mucosa of neonatal rats. A: representative Western blot. B: relative density of GLUT5 protein. Bars are means ± SE (n = 3). Density of GLUT5 protein in glucose-perfused (control) intestine was designated as 100%, and the densities in other groups were normalized to this value. Different letters indicate a significant difference in transporter abundance.

 
The twofold increase in fructose uptake rate induced by fructose solutions (P < 0.01) was consistent with the increase in GLUT5 protein abundance in the BBMV (Fig. 11). SH-5 prevented the fructose-induced increase in fructose uptake rate (P < 0.01). Glucose uptake rate was not affected by SH-5.



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  		Fig. 11. SH-5 (10 nM), an inhibitor of PI3-kinase, blocked the fructose-induced increase in fructose uptake, but did not affect glucose uptake, in the small intestine of neonatal rats. Bars are means ± SE (n = 5–6). Uptake rate in glucose-perfused (control) rats was designated as 100%, and the uptake rates in other rats were normalized to this value. Different letters indicate a significant difference in uptake rates.

 
Effect of EGF, an activator of PI3-kinase, on sugar uptake and GLUT5 mRNA expression. EGF markedly increased fructose, but not glucose, uptake rate in the small intestine (P < 0.01) (Fig. 12). Like the effect of wortmannin on fructose uptake rate, the EGF-induced fructose uptake rate was inhibited by wortmannin (P < 0.01). GLUT5, SGLT1, and GLUT2 mRNA abundance was not affected by EGF and wortmannin (data not shown).



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  		Fig. 12. EGF (60 ng/ml), an activator of PI3-kinase, increased fructose uptake rate in the small intestine of neonatal rats. Wortmannin (10 nM) blocked the EGF-stimulated increase in fructose uptake. Glucose uptake rate was not affected by EGF and Wortmannin. Bars are means ± SE (n = 5–6). Uptake rate in KRB-perfused rats was designated as 100%, and the uptake rates in other rats were normalized to this value. Different letters indicate a significant difference in uptake rates.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The intestine of neonatal rats normally does not encounter fructose in significant amounts until after completion of weaning when weaned pups subsist entirely on feed obtained from their habitat. At completion of weaning, endogenous (hard-wired) mechanisms trigger marked increases in intestinal fructose transport (48). If pups were fed (10) or the intestine perfused (27) with fructose before completion of weaning, intestinal fructose transport would increase. In the wild, this ability to enhance fructose transport (and activities of many digestive enzymes) before completion of weaning may allow pups to survive sudden loss of the mother (24). Induction of intestinal fructose transport occurs rapidly after introduction of luminal fructose and is normally preceded by marked increases in GLUT5 mRNA and protein abundance (27). In this paper, we found that increases in fructose transport rate and GLUT5 protein abundance can be modulated independently of changes in GLUT5 mRNA level, because inhibitors of PI3-kinase and Akt have clearly prevented the fructose-mediated increases in fructose transport and GLUT5 protein abundance without affecting GLUT5 mRNA levels. This indicates that the synthesis of GLUT5 transporter and its insertion into the brush-border membrane can be disrupted by inhibiting the PI3-kinase/Akt pathway.

PI3-kinase and intestinal fructose uptake. Wortmannin at 10 nM and LY-294002 at 100 nM are, at these low concentrations, highly specific inhibitors of PI3-kinase (52). Wortmannin binds covalently to the PI3-kinase catalytic subunits at the wortmannin binding site, whereas LY-294002 binds competitively to the ATP site (49). Despite differences in their sites of action on PI3-kinase, similarities in wortmannin and LY-294002 inhibition of GLUT5 indicate a likely involvement of PI3-kinase in enhancement of intestinal fructose transport during development. Immunocytochemistry results also suggest that the fructose-induced increase in GLUT5 protein might be related to the increase in PIP3.

To provide additional evidence regarding the link between PI3-kinase and induction of fructose transport, EGF was perfused in the intestine to stimulate the activity of PI3-kinase. EGF, like luminal fructose, significantly increased fructose uptake, and wortmannin inhibited the EGF-induced increase in fructose uptake. These results implied that PI3-kinase was activated by EGF, and PI3-kinase activation then stimulated fructose transport in the enterocytes. PI3-kinase-modulated effects on fructose metabolism have also been demonstrated in fibroblasts and hepatoma cells in which LY-294002 was shown to inhibit 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase expression by EGF (15). Fructose perfusion has also been shown to stimulate phosphofructo-2-kinase/fructose-2,6-bisphosphatase transcription in neonatal rat intestine (9).

We also evaluated the effect of PI3-kinase/Akt inhibitors on glucose uptake to understand whether or not their effect on fructose uptake is specific. Consistent with previous studies (16) in neonatal rat intestine, glucose uptake is not influenced by fructose perfusion. Although intestinal glucose uptake in adult rats can be regulated by dietary carbohydrate (48), it may be independent of dietary or luminal carbohydrate in neonates because the types of nutrients and "dietary" (milk) concentrations of these nutrients do not vary. In this study, glucose perfusion plus wortmannin, LY-249002, or SH-5 did not affect glucose uptake, indicating that the activity of SGLT1 is not affected by these inhibitors and that the PI3-kinase/Akt pathway specifically inhibits the fructose-induced fructose uptake. The finding that PI3-kinase did not modulate glucose uptake is reasonable because PI3-kinase inhibitors affect glucose uptake only when insulin is involved. For example, in cultured adipocytes, wortmannin did not inhibit baseline glucose uptake (6) but when these adipocytes were incubated with insulin, wortmannin blocked the insulin-induced glucose uptake.

PI3-kinase and GLUT5. Parallel twofold (from several sets of experiments) increases in fructose-induced fructose uptake rate and in GLUT5 transporter abundance in the brush-border membrane indicate that uptake rates are not due to significant increases in GLUT5 activity, but instead either to increases in GLUT5 protein synthesis or in translocation of the GLUT5 protein from intracellular stores. Our data cannot distinguish between these two mechanisms, because GLUT5 in homogenates also increased by about twofold in fructose-perfused intestine.

There has been no other study on PI3-kinase and fructose in the small intestine. However, insulin treatment of cultured skeletal muscle stimulates parallel increases in fructose uptake rate and GLUT5 protein abundance (23). Because PI3-kinase modulates transport by GLUT4 translocation in skeletal muscle, it is possible that PI3-kinase also regulates GLUT5 in a similar manner in muscle and other insulin-responsive tissues like the intestine.

PI3-kinase plays a major role in the regulation of plasma glucose concentrations by mediating the wortmannin-inhibitable transfer of GLUTs, specifically the muscle-fat facilitative glucose transporter isoform GLUT4, from intracellular compartments to the plasma membrane. In muscle (40) and adipocytes (41), it has long been known that insulin-stimulated translocation of GLUT4 from intracellular stores to the cell surface involves signaling pathways that are dependent on PI3-kinase. The transfer of GLUT4 is dependent on actin filament remodeling, and the participation of the downstream PI3-kinase-effector Akt can be demonstrated by Akt presence in the remodeled actin (40). The contribution of Akt to GLUT translocation in adipocytes can readily be confirmed because simultaneous increases in Akt kinase activity, Akt phosphorylation, and GLUT4 translocation can be inhibited by an Akt inhibitor, ML-9, or in cells transfected with a dominant negative form of Akt (25). In this study, we have also demonstrated the participation of Akt toward fructose-induced increases in fructose absorption as well as in GLUT5 transporter abundance in the intestinal brush-border membrane.

There have also been reports of wortmannin-sensitive and therefore PI3-kinase-dependent translocation of GLUT1, the erhythrocyte isoform, to the plasma membrane of skeletal muscle cells (32). Hence, the role of PI3-kinase and Akt may not only be limited to GLUT4 but also apply to other GLUTs, and the mechanism underlying the effect of PI3-kinase on intestinal fructose transport may be via inhibition of GLUT5 insertion into the brush-border membrane.

PI3-kinase and other transport systems. PI3-kinase modulates the translocation not only of GLUTs but also of other transport systems. It is involved in the insulin-stimulated translocation of the fatty acid transporter FAT/CD36 from an intracellular depot to the plasma membrane of rat skeletal muscle (34). Recent work also determined that PI3-kinase promotes the translocation of calcium (50) and transient receptor potential channels (2) in cultured neurons.

An interesting parallelism and potential cross talk between signaling pathways can be demonstrated regarding regulation of GLUT5 in the neonatal rat intestine and the Na+-taurocholate-cotransporting polypeptide (NTCP) in the rat liver. cAMP stimulates hepatocyte taurocholate (53) and intestinal fructose (8) transport in rats. It turns out that the cAMP-stimulated increases in taurocholate transport can be prevented by PI3-kinase and Akt inhibitors and involves the translocation of NTCP transporters to the plasma membrane of hepatocytes (35). Although we did not use PI3-kinase and PKB inhibitors in our cAMP experiments (8), it is interesting to note that the fructose-induced fructose transport in rat neonates that can be inhibited by dideoxyadenosine (an inhibitor of adenylyl cyclase) in previous work has also been shown to be inhibited by PI3-kinase and Akt inhibitors.

Effect of fructose on PI3-kinase. Although fructose had no effect on the mRNA expression of the regulatory and catalytic subunits of PI3 kinase, it may have an effect on PI3-kinase activity. Fructose increases the activity of PI3-kinase in skeletal muscle (3), which in turn increases production of PIP3. The PI3-kinase pathway can control GLUT4 insertion into the plasma membrane by relying on effector systems that respond to changes in amplitude of PIP3 signals (47). We found that PIP3, unlike brush-border GLUT5, was localized mainly in the cytosol of the epithelial cells. However, like GLUT5, PIP3 staining seemed much stronger in the fructose- compared with glucose-perfused intestine. These increases in GLUT5 are shown immunocytochemically to take place in the same mid- to upper-villus cells as those with apparently a higher concentration of PIP3. The matching increases in GLUT5 and PIP3 staining suggest that the GLUT5 substrate fructose may modulate PI3-kinase activity, which in turn regulates GLUT5 synthesis or translocation.

Role of Akt. Although there are many downstream effectors regulated by PI3-kinase, in this study, we focused on Akt to verify its effect on the fructose-induced increase in fructose uptake in the small intestine. SH-5 is a phosphatidylinositol analog that selectively inhibits the activation of Akt by decreasing its phosphorylation without affecting the phosphorylation of PDK-1 and other downstream kinases. In the present study, we found that the SH-5 markedly blocked the fructose-induced increase in fructose uptake suggesting that Akt may regulate fructose absorption in the small intestine. There has been no study on fructose metabolism and Akt phosphorylation in the small intestine, but in hyperinsulinemic, hypertensive rats fed fructose and not treated with insulin, which can increase Akt activity via PI3-kinase, basal Akt activity was elevated about twofold in skeletal muscle (3). High levels of a fructose metabolite and glycolysis modulator fructose-2,6-bisphosphate in the liver also increases phosphorylation of Akt (55).

Cellular localization of GLUT5 and GLUT2 transporters. Our immunocytochemical and Western blots indicate that GLUT5 is localized mainly in the brush-border membrane of fructose- and glucose-perfused intestines. These findings are consistent with previous work in adult rat and human (11, 26, 42). The apparent increase in GLUT5 staining in the brush-border membrane of fructose-perfused intestine was validated with an increase in GLUT5 protein abundance detected by Western blotting of BBMVs.

The localization of GLUT2 in enterocyte membranes is more controversial than that of GLUT5, and a detailed discussion is beyond the scope of this paper. The classical model of transepithelial sugar transport involves SGLT1 and GLUT5 in the brush border and GLUT2 in the basolateral membrane, and their coordinated, combined activities are sufficient to account for intestinal absorptive capacities (16, 17). Because kinetic studies of sugar absorption suggest that there may be a second glucose transporter in the brush-border membrane or even a different type of transport process, explanations that deviate from the classical model have been suggested, among them the solvent drag (38) and the brush-border GLUT2 (30) models. The latter hypothesis suggests that primarily under conditions of excess luminal glucose or fructose loads, GLUT2 transporters are transiently and rapidly translocated to the brush-border membrane. In GLUT2-null mice, fructose uptake in brush-border membrane vesicles was half that of wild-type mice, suggesting that brush-border GLUT2 absorbed 50% of luminal fructose (21). It is interesting to note that GLUT2-null mice show a normal glucose tolerance test indicating normal brush-border and basolateral glucose transport in the GLUT2-less intestine. Another glucose transporter, GLUT7, which is quite similar in AA sequence to that of GLUT5, was reported to be expressed primarily in the intestinal brush border (33). A significant presence of facilitative, glucose-selective GLUT transporters in the brush-border membrane may be problematic, however, because it facilitates the efflux across the brush-border membrane of glucose accumulated via SGLT1 at high concentrations in the cytosol (54). In contrast to findings of brush-border GLUT2 in perfused intestines of adult rats, we found vanishingly small amounts of GLUT2, relative to GLUT5 concentrations, in the brush-border membrane of neonatal rat intestines perfused in vivo with either 100 mM fructose or 100 mM glucose for 4 h. In fact, there were more GLUT2 transporters in tissue homogenates compared with those in BBMVs of the same samples. GLUT2 also could not be found in the brush-border membrane of normal and diabetic humans (13).

Regulation of intestinal sugar transport may be tightly linked to development, and this linkage may explain the apparently contradictory findings. For example, SGLT1 is regulated by dietary carbohydrates in adults but not in weaning rats. Because GLUT2 expression in rat pups was not inducible with luminal glucose perfusion, our findings may also represent an equally interesting lack of response by GLUT2 in the neonatal rat intestine.

Whereas brush-border fructose uptake rate and brush-border membrane as well as homogenate concentrations of GLUT5 increased significantly in the fructose-perfused compared with those in the glucose-perfused intestine, there was no such correlation between changes in fructose transport and in GLUT2 protein abundance in the brush-border and homogenates. Hence, intestinal fructose transport was mainly mediated by its own transporter GLUT5, which in turn was upregulated by its own substrate fructose in the lumen.

In conclusion, PI3-kinase/Akt signaling pathway regulates the fructose-induced GLUT5 activity and protein synthesis, but does not participate in the fructose-stimulated GLUT5 mRNA expression in the neonatal rat small intestine (Fig. 13).



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  		Fig. 13. PI3-kinase/Akt signal transduction pathway may be involved in the regulation of fructose uptake and GLUT5 protein synthesis or translocation but not in GLUT5 gene transcription.

 

   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Science Foundation Grants IBN-998808 and 235011 as well as United States Dept. of Agriculture–Cooperative State Research, Education, and Extension Service–National Research Initiative. Grants 2004-35206-14154 and 2003-35102-13520.


   ACKNOWLEDGMENTS
 
We thank Drs. C. Burant and M. Lee for cDNA probes and Dr. L. Gaspers for primary hepatocytes.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. P. Ferraris, Dept. of Pharmacology and Physiology, MSB H621, New Jersey Medical School, 185 S. Orange Ave., Newark, NJ 07103

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.


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