1 Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey (UMDNJ)-New Jersey Medical School, Newark 07103-2714
2 Center for Applied Genomics, Public Health Research Institute, Newark 07103-3506
3 Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, New Jersey 07103-2714
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ABSTRACT |
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gluconeogenesis; glucose-6-phosphatase; GLUT5; regulation; glucose-6-phosphate translocase
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INTRODUCTION |
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There have been only five microarray studies on mammalian small intestine, and there is as yet no microarray study on the response of the small intestine to a nutrient signal. This is surprising, since the small intestine constitutes the first barrier to nutrient metabolism in all mammals and is therefore strategically positioned to provide the major regulatory mechanism(s) that may be altered by nutrient availability. There has been one study on the effect of protein quantity and quality on rat hepatic gene expression, but the RNA from several rats was pooled so that results were based on only a single microarray (12). In this study, we found many genes along with GLUT5 to change in expression after a 4-h perfusion with fructose, indicating that many signaling and metabolic pathways in the small intestine are responsive to the presence of fructose in the lumen. To determine potential signals that may increase shortly after initiation of fructose perfusion, we also determined gene expression in rat intestines perfused for only 20 min. Here, we found a smaller number of genes whose expression changes with fructose perfusion.
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MATERIALS AND METHODS |
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For the microarray study, we chose to perfuse fructose, because it was the only nutrient known to stimulate GLUT5, and it did not affect the mRNA expression of other brush border sugar transporters. Glucose perfusion was an excellent control, because it did not affect GLUT5 expression and activity.
In a subsequent experiment to determine the regulation of glucose-6-phosphatase (G6P) expression, rat intestines were perfused with HF, HG, 100 mM -methylglucose (a nonmetabolizable, SGLT1-transportable glucose analog; Ref. 11) in Ringer and normal Ringer alone. The osmolalities of all perfusion solutions were the same (290 mosmol/kgH2O), with fructose, glucose, and
-methylglucose replacing NaCl (see section on perfusion model for Ringer composition).
Although GLUT5 expression is known to peak at 4 h after HF perfusion, other intestinal genes may respond at a shorter time intervals. The choice of 20-min perfusion was based on a previous study indicating that the mRNA and protein expression of the immediate-early genes c-fos and c-jun in epithelial cells peaked at this duration when the intestine was perfused with either glucose or fructose (21).
An everted sleeve method was employed for determining glucose and fructose uptake rates. Relative quantitative reverse transcription-polymerase chain reaction (RT-PCR) was initially used to examine GLUT5 mRNA abundance in perfused intestinal tissues, to ensure that tissues used for subsequent (and labor-intensive) microarray experiments responded in a manner similar to tissues in previous work. An oligonucleotide microarray was used for screening the genes that were affected by fructose and glucose perfusion. Finally, some of the up- and downregulated genes were confirmed by semiquantitative RT-PCR.
Animals
Adult male and female Sprague-Dawley rats weighing 200 g were purchased from Taconic (Germantown, NY) and bred. Rats were housed in a temperature-controlled room (2224°C) with a 12:12-h light-dark cycle in the research animal facility, and allowed access to water and food ad libitum (Purina Mills, Richmond, IN). 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. Midweaning rat pups were used in this study. The pups were kept with their dams until they were 2022 days old, and then they were randomly selected according to experimental needs. All the procedures conducted in this study were approved by the Institutional Animal Care and Use Committee, UMDNJ-New Jersey Medical School.
Perfusion Model
Rat intestinal perfusion was conducted following the method of Jiang and Ferraris (20). Briefly, rat pups (2022 days old, not starved) were anesthetized by intraperitoneal injection of urethane (1.4 g/kg body wt). After its contents were flushed, the small intestine was continuously perfused with solution (100 mM glucose or fructose in Ringer) at a rate of 60 ml/h at 37°C using a peristaltic pump. The composition of the perfusion solution was (in mM) 78 NaCl, 4.7 KCl, 2.5 CaCl2·H2O, 1.2 MgSO4, 19 NaHCO3, 2.2 KH2PO4, and 100 sugar. Perfusion for the microarray experiment was performed in sets of two (one perfused with HG, another with HF). Perfusion for the G6P experiment (see Fig. 5) was done in sets of four (normal Ringer, -methylglucose, fructose, glucose) parallel perfusions. Rats perfused within a set were littermates subjected to exactly the same conditions except for differences in perfusion solution.
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Total RNA Extraction
The total RNA was extracted from 100 mg tissue (stored in 80°C) using RNeasy Midi kits (Qiagen, Valencia, CA) according to the manufacturers instructions. The RNA sample was resuspended in RNase-free water and stored at 80°C in a freezer for the subsequent RT-PCR, Northern blot, and microarray analysis.
Microarray
The design, analysis, and interpretation of this microarray experiment followed in general the suggestions of other microarray studies (24, 33, 37). The detailed protocols and primary data from this study are available at the Gene Expression Omnibus database (GEO; http:/www.ncbi.nlm.nih.gov/geo), under accession number GSE996.
Since spotted microarrays allowed only two treatments to be compared directly, we were constrained to use glucose as control not only because its perfusion (20) and consumption (8) had no effect on the gene of interest GLUT5, but also because of the following reasons. Unperfused controls were not useful since intestinal perfusion even by a nonmetabolizable, nonabsorbable sugar such as mannitol triggered marked responses by many genes in the small intestine (21). Perfusion with a nonmetabolizable sugar as control also had a problem with distinguishing fructose-response genes from numerous metabolic-response genes. In fact, many genes were nonspecifically regulated by starvation and sugar metabolism (39).
Because our microarray did not contain oligonucleotides representing the entire rat genome, the list of genes was far from complete but nonetheless identified for the first time genes responsive to luminal fructose. Expression of these genes must be specifically and directly or indirectly enhanced by luminal fructose because we used as control intestines perfused with glucose, eliminating from consideration the genes that responded nonspecifically to luminal sugars. Conversely, by using fructose as the reference, readers interested in glucose metabolism can interpret the downregulated genes as genes that were upregulated with glucose perfusion.
Array design description.
We used a rat 5,000-oligonucleotide array [Rat 5K Oligo Array (NGEL 2.0.1); Center for Applied Genomics, PHRI, Newark, NJ]. A detailed description of array design elements is available from the following web sites: http://www.cag.icph.org and http://www.labonweb.com. The array is built from 4,854 oligonucleotides representing 4,803 independent genes. All oligonucleotides are 6070 nucleotides in length. Oligonucleotides were printed in 3x SSC at a concentration of 40 µM on poly-L-lysine-coated glass slides (http://www.microarrays.org) using a GeneMachines OmniGrid 100 microarrayer (Genomic Solutions, Ann Arbor, MI). Arrays were stored at room temperature in a desiccator until use.
Labeling and hybridization procedures and parameters.
The 3DNA Submicro Oligo Expression Array Detection Kit (Genisphere, Hatfield, PA) was employed to synthesize and label the complementary DNA (cDNA) with fluorescent dyes Cy3 or Cy5. The indirect labeling method was chosen because it is more sensitive and consistent than the direct labeling method or other indirect techniques (38). In the preliminary experiments, 3, 4, and 5 µg of total RNA was tested for labeling and hybridization conditions. In the actual experiments, 4 µg of total RNA from each sample was used for labeling and hybridization, because at this amount of total RNA, the hybridization signal-to-noise ratio was high, signals from almost all modestly expressed genes were detectable, and signals from highly expressed genes were not fully saturated.
Experiment description.
In the first experiment, rat intestines were perfused with HF (n = 5) or HG (n = 5) for 4 h. RNA from fructose-perfused intestines was labeled with Cy5, and RNA from glucose-perfused intestines with Cy3. To eliminate dye-bias, one randomly selected fructose-perfused intestine was labeled in the opposite manner with Cy3, as was the corresponding glucose-perfused intestine with Cy5. Results from this dye-flip were used to correct results as described later. In the 4-h perfusion experiment, we decided to label one-way only and eliminated dye bias with a dye-flip experiment because we had an odd number of paired samples, and the number of Cy3-labeled fructose-perfused intestines would never be the same as that of Cy3-labeled glucose-perfused intestines.
In the second experiment, rat intestines were perfused with HF (n = 4) or HG (n = 4) for 20 min. RNA from two HF-perfused intestines was labeled with Cy5, and their corresponding HG-perfused intestines were labeled with Cy3. Conversely, RNA from another two HF intestines was labeled with Cy3, and their corresponding HG intestines were labeled with Cy5.
To identify additional potentially false-positive genes, three randomly chosen samples from HF- and HG-perfused intestines were used for "self-against-self" experiments, whereby RNA from each sample was labeled with both dyes. Since the dye-labeled samples were identical, these hybridizations should ideally produce similar intensities for every spot for both Cy3 and Cy5 channels. Under these conditions, no gene should be over- or underexpressed, and spots exhibiting significant differences in intensity between the two channels were eliminated from consideration (see Data processing below). Finally, two "capture-primer" experiments were performed where Cy3 and Cy5 capture sequences with no sample RNA were loaded onto the array. Since the capture sequences should not correspond to any of the oligonucleotide sequences on the array, ideally, there should be no fluorescence for each microarray spot for both Cy3 and Cy5 channels.
Data processing.
Slides were scanned for Cy3 and Cy5 fluorescence using an Axon GenePix 4000A microarray reader (Axon instruments, Union City, CA). The density of the spots was detected using GenePix 4.0 (Axon Instruments). When measuring the density of the spots, the ratio of Cy3 and Cy5 dyes was adjusted to 1 as a global normalization for each sample slide. The results (median pixel density of each array spot was used) were normalized by LOWESS (locally weighed scatter smoother) method so that the results can be compared among the experiments performed at different times (37).
The following rules were made to eliminate genes that were considered false positives and to choose genes that might change significantly with fructose perfusion. First, genes in array spots with a density over twofold that of background in the 3DNA capture primer experiment were eliminated, because these genes should not fluoresce above background, as no sample RNA was added. Second, in the actual array experiments, genes whose expression was so low that their average median density was less than twofold that of background density were also eliminated (23). Third, genes whose expression changed by >50% in any one of three self-self hybridization experiments were eliminated, because these genes should not change in expression when Cy3 and Cy5 labeled the same sample. Fourth, nutrient-induced changes in gene expression should not be altered when a different dye was used to label the same sample. To minimize dye bias, any gene whose expression changed from a positive value (in this study, defined as gene expression was greater in HF- over HG-perfused intestine) to a negative value when the dye was switched was eliminated. Conversely, any genes whose expression changed from a negative to a positive value (gene expression greater in HG- over HF-perfused intestine) when the dye was switched were eliminated. Fifth, only genes that changed by 50% in at least three of five comparisons in the 4-h perfusion experiment, and in two of four comparisons in the 20-min perfusion experiment, were considered. Although genes whose expression changed markedly (>>50%) with sugar perfusion might initially be more interesting, we chose a 50% change as guide, because we also wanted to examine genes whose expression might change moderately, but consistently, with HF (or HG) perfusion.
RT-PCR
A OneStep RT-PCR kit (Qiagen) was used in this experiment to verify changes in expression as detected by microarray analysis. Briefly, the reaction was performed on a thermal cycler (Techne, Duxford, Cambridge, UK) for 30 min at 50°C, 15 min at 95°C for reverse transcription reaction and activation of Taq DNA polymerase. The DNA amplification procedure was set at 45 s, 94°C (denature); 45 s, 6065°C (annealing); and 90 s, 72°C (extension) for a total number of cycles optimal (see next paragraph) for PCR amplification of different genes. Pictures of the gels were taken using a CCD camera for determination of the density of the bands by a densitometer (FluorChem 8800; Alpha Innotech, San Leandro, CA). 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 (16, 26, 35).
Determination of Optimal PCR Cycles for Each Gene
The number of PCR cycles optimal for determining relative changes in mRNA abundance varied. An exponential relationship between RT-PCR products (represented by ethidium bromide density) and DNA amplification cycles was demonstrated between 21 and 33 cycles for GLUT5, 21 and 27 cycles for NaPi2, and 18 and 27 cycles for GAPDH. The optimal number was considered to be the midpoint of the exponential phase, or 27 cycles for GLUT5, 24 for NaPi2, and 23 for GAPDH. We measured the optimal number of cycles for 20 candidate genes whose mRNA abundance were confirmed using semiquantitative RT-PCR (Table 1).
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Statistical Analysis
Data are presented as means ± SE. Paired student t-test was used to determine perfusion-related differences in fructose and glucose uptake, and in the relative amounts of gene expression in RT-PCR experiments using log10 of the ratio of the target gene to the housekeeping gene, GAPDH. A one-sample t-test was used to determine significance of changes in gene expression in the microarray results. The values tested were derived from the GenePix algorithm M = log2(R/G), where R = red channel signal = F635; G = green channel signal = B532, and the hypothetical mean was 0 (meaning no change between HF and HG). For consistency with the microarray results, the uptake rates and gene expression results from RT-PCR were converted to ratios of HF to HG. Concordance between the results of RT-PCR and the microarrays was evaluated by correlation analysis. P < 0.05 was considered statistically significant. Statistical analysis was conducted using the StatView program (Abacus Concepts, Berkeley, CA).
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RESULTS |
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Comparison of Microarray and RT-PCR Results
There were 24 genes whose mRNA abundance was reanalyzed by RT-PCR, and in 80% of these genes, the results from RT-PCR matched those from the microarray experiments (Table 4). The slope of the line between microarray and RT-PCR results was not significantly different from unity, indicating that after appropriate correction for false positives as detected by capture primer and dye-flip experiments, changes in mRNA abundance as detected by microarray will likely be similar to those detected by RT-PCR. Based on a correlation analysis, r2 was 0.843 (P < 0.0001) indicating the general reliability of the results obtained from the microarray. Genes that did not change in expression by microarray were also found to be unchanged when evaluated by RT-PCR (Table 4C).
G6P Expression Increases with HF
Since microarray results were expressed in ratios of RNA abundance in HF- relative to HG-perfused intestine, we determined whether high ratios of G6P expression increased with HF and/or decreased with HG perfusion by comparing mRNA abundance among HG-, HF-, -methylglucose-, and normal Ringer-perfused intestines. G6P mRNA abundance was markedly greater in HF-perfused intestine compared with normal Ringer-perfused (P < 0.01) and
-methylglucose-perfused (P < 0.001) intestines (Fig. 5), paralleling results obtained with GLUT5 (7). G6P mRNA abundance was the same (P > 0.05) among HG-, normal Ringer-, and
-methylglucose-perfused intestines.
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DISCUSSION |
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Genes Upregulated with Luminal Fructose
Genes involved in sugar metabolism.
Ten of 24 upregulated genes at 4 h of fructose perfusion are either directly involved in enzymatic metabolic reactions (Table 2) or regulate those reactions. Of these 10 upregulated genes, 6 metabolize sugars, and 5 of the 6 add or remove phosphate groups. These six genes participate in fructose and mannose metabolism, glycolysis/gluconeogenesis, pentose phosphate pathway, and galactose metabolism. One gene, 6-phosphofructo 1-kinase, catalyzes the formation of fructose-1,6-bisphosphate from fructose-6-phosphate with hydrolysis of ATP and participates directly in all four of these metabolic pathways (Kyoto Encyclopedia of Genes and Genomes, http://www.genome.ad.jp/kegg/). Another gene, fructose-1,6-bisphosphatase catalyzes the opposite reaction (hydrolysis of fructose-1,6-bisphosphate to produce fructose-6-phosphate and inorganic phosphate) and is thought to participate in fructose and mannose, gluconeogenic, and pentose phosphate metabolic pathways.
The one gene that is upregulated and is unique to the fructose and mannose metabolic pathway is fructose-2,6-bisphosphatase (Table 2), which hydrolyzes fructose-2,6-bisphosphate to fructose-6-phosphate and orthophosphate. This same enzyme, when modulated by a cAMP-dependent protein kinase, becomes 6-phosphofructo 2-kinase, which catalyzes the opposite reaction (synthesis of fructose-2,6-bisphosphate from fructose-6-phosphate and ATP). It is interesting to note that dibutryl-cAMP, a permeable nonmetabolizable analog of cAMP, can significantly alter intestinal fructose transport without changing GLUT5 mRNA abundance (Cui et al., unpublished observations). GLUT5 is a facilitative transporter, and whenever free fructose concentration is decreased in the cytosol, it will result in increased downhill gradient for fructose transport into the cell. Hence, conditions that favor intracellular fructose metabolism may alter fructose uptake rate.
Fructose-2,6-bisphosphate is an allosteric inhibitor of fructose-1,6-bisphosphatase (Fig. 6). Since fructose-2,6-bisphosphatase is overexpressed during fructose perfusion, removal of fructose-2,6-bisphosphate may in turn remove the inhibition of fructose-1,6-bisphosphatase, thereby enhancing gluconeogenesis. Significant gluconeogenesis is now known to occur in the small intestinal epithelial cells, which can supply up to a quarter of total glucose production during insulinopenia (6).
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The two other genes in the glycolytic/gluconeogenic pathway clearly increasing with fructose perfusion are G6P catalyzing the hydrolysis of glucose-6-phosphate to glucose and orthophosphate as well as phosphoglycerate mutase, an isomerase which converts 2-phospho-D-glycerate, a precursor of phosphoenolpyruvate, to 3-phospho-D-glycerate (Table 2). Changes in phosphoenolpyruvate concentrations may affect cellular levels of pyruvate, which is a major metabolite whose concentration is pivotal in regulating many metabolic pathways. Expression of pyruvate dehydrogenase kinase 2, which inactivates pyruvate dehydrogenase (enzyme complex converting pyruvate to acetyl-CoA) also increases with fructose perfusion. This means that fructose perfusion may inhibit the conversion of pyruvate to acetyl-CoA, an inhibition consistent with the increase in expression of gluconeogenic enzymes. Acetyl-CoA is an important intermediate in lipid metabolism and in oxidative phosphorylation.
Of all the metabolic enzymes discussed above, only the expression of G6P increases after just 20 min of HF perfusion (Table 5A), well before GLUT5 mRNA abundance increases. G6P mRNA abundance increases from about twofold greater than HG perfusion at 20 min to fourfold greater than HG perfusion after 4 h. This indicates that luminal fructose may trigger transcription of G6P even more rapidly than it triggers GLUT5 transcription. It is also intriguing to note that the mRNA abundance of G6P seems highly sensitive to luminal fructose and not to luminal glucose (Fig. 5). This pattern is much like that of GLUT5 but not that of SGLT1 (28) and GLUT2 (7). The equilibrium concentrations of fructose-6-phosphate:glucose-6-phosphate lie sevenfold toward glucose-6-phosphate, and may suggest why glucose loading has little effect on fructose-responsive enzymes.
G6P is part of a system consisting of four subunits found in the membrane of the endoplasmic reticulum (34). Not only does the expression of G6P (the hydrolase or catalytic subunit) increase with fructose perfusion, but also increased is the expression of a second subunit, glycogen storage disease type 1b, also known as the G6P translocase (Table 2). These parallel increases highlight the importance of the entire G6P system to intestinal fructose metabolism, and suggest a linkage to GLUT5 regulation.
Membrane proteins.
Expression of GLUT5 and GLUT4 (Table 2), the facilitative glucose transporter in skeletal and smooth muscle and in adipose tissue, also increases. Either GLUT4 mRNA is also found in intestinal epithelial cells, or GLUT4 expression increased in smooth muscle tissue found in the submucosal and muscularis layers of the small intestine. The role of other membrane transporters or proteins that regulate those transporters is not clear.
Development, transcription factor, and other genes.
Whereas luminal fructose can stimulate GLUT5 expression in weaning (15 days) rats, it has no effect on GLUT5 expression in younger rats (19). Hence, the ability of fructose to stimulate GLUT5 transcription is modulated by development. There are two fructose-upregulated genes that are also strongly involved in regulating development: glypican 3 and activin receptor-like kinase (Table 2). Glypican 3 is involved in controlling the growth of embryonic mesodermal tissues, and in regulating the shape of intestinal epithelial cells during morphogenesis (5). In contrast, activin receptor kinase is not well characterized but belongs to a novel subclass of cell-surface receptors with predicted serine/threonine kinase activity. These receptors regulate activins, which belong to the TGFß family, and are involved in the patterning of the mesoderm like glypican 3 (2).
Activating transcription factor 4 and zinc finger protein are factors likely involved in upregulating transcription of genes turned on by fructose perfusion (Table 2). In hepatocytes of aged rats, GADD153 (also known as C/EBP homologous protein, or CHOP) expression is induced by epidermal growth factor (EGF) (18). Perfusion of EGF in neonatal rat intestine, specifically enhances fructose transport (Cerquiera, Cui, and Ferraris, unpublished observations). The role of other remaining genes is not known.
Genes Downregulated with Luminal Fructose
A relatively large number of genes is significantly downregulated with fructose perfusion, but it is not clear how these are specifically related to fructose induction of GLUT5. Fructose perfusion inhibits the expression of cytochrome-c oxidase, an electron carrier in the respiratory chain (Table 3). Inhibition of a key component of oxidative phosphorylation is consistent with increased expression of gluconeogenic enzymes discussed previously. Since acetyl-CoA is not catabolized when oxidative phosphorylation is inhibited, its concentration increases. Increases in acetyl-CoA stimulate the initial steps of gluconeogenesis. The gluconeogenic pathway should dramatically increase intracellular inorganic phosphate concentrations, which in turn, should downregulate transporters that take in inorganic phosphates from the intestinal lumen or blood. Indeed, fructose perfusion consistently and markedly downregulates the expression of NaPi2b (Table 3), the sodium-dependent phosphate cotransporter that absorbs inorganic phosphates from the intestinal lumen into the cytosol. This suggests that NaPi2b, known to be regulated by dietary phosphate levels (30), may also be modulated by intracellular phosphate concentrations.
The role of the sodium-myo-inositol transporter SMIT (Table 3) is not clear, because it has not previously been reported in the intestine. SMIT is a member of SLC5, the gene family of sodium-dependent glucose cotransporters (36), and SGLT1 is known to increase with luminal glucose in adult rodent intestines (14). GLUT2 is the intestinal basolateral glucose and fructose transporter (13), and its expression may increase with glucose perfusion. GLUT2 expression is regulated by both luminal fructose and glucose (7), but glucose is often reported as a more important regulator (32).
The endothelin receptor (Table 3) is involved in development, and a mutation in this gene results in intestinal hypertrophy (17). Likewise, the catenins are abundant cellular proteins, and their spatial distribution is critical in establishing tissue patterns during embryonic development.
TGFß (Table 5B) typically inhibits cell proliferation and is found in high levels in both rat milk and neonatal rat small intestine, and endogenous production in the intestine increases during the weaning period (40). Inhibiting the expression of this factor may stimulate proliferation.
Future Studies
Changes in gene expression may arise not only from various cell types in the submucosal, muscle, and serosal layers but also from other cell types in the mucosal layer itself; hence, it should be established that these changes occur in the enterocytes where GLUT5 is found. However, changes in gene expression are likely to be events occurring mainly in the enterocytes because only the mucosal epithelium was exposed to and can absorb luminal fructose and glucose. The mucosal epithelium consists mainly of absorptive enterocytes (1), and we showed by in situ hybridization (21) as well as immunocytochemistry (21) that mRNA and protein expression of GLUT5 and the transcription factors c-fos and c-jun increase only in enterocytes of perfused intestines. Ongoing studies also suggest that phosphatidylinositol-3,4,5-triphosphate, a second messenger in the PI3-kinase system, concentrations also increase only in enterocytes whose GLUT5 mRNA abundance increases with HF perfusion.
The biggest challenge is to distinguish which of the genes specifically modulate the effect of luminal fructose on its transporter GLUT5 and which of the genes are responding secondarily to fructose perfusion. Fortunately, a cluster of genes involved in glycolysis and gluconeogenesis stand out as candidate genes regulating GLUT5 expression, and studies using gluconeogenic inhibitors are warranted, to assess the role of some gluconeogenic enzymes such as G6P on GLUT5 regulation.
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GRANTS |
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ACKNOWLEDGMENTS |
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Present address of P. Tolias: Ortho-clinical Diagnostics-a Johnson and Johnson Co., 1001 US Hwy 202, Raritan, NJ 08869.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. P. Ferraris, Dept. of Pharmacology and Physiology, UMDNJ-New Jersey Medical School, Newark, NJ 07103-2714 (E-mail: ferraris{at}umdnj.edu).
10.1152/physiolgenomics.00056.2004
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REFERENCES |
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