The FASEB Journal article fj.15-272195. Published online June 12, 2015.

The FASEB Journal • Research Communication

Transport, metabolism, and endosomal traffickingdependent regulation of intestinal fructose absorption Chirag Patel,* Veronique Douard,*,1 Shiyan Yu,† Nan Gao,† and Ronaldo P. Ferraris*,2 *Department of Pharmacology and Physiology, New Jersey Medical School, and †Department of Biological Sciences, School of Arts and Sciences, Rutgers University, Newark, New Jersey, USA Dietary fructose that is linked to metabolic abnormalities can up-regulate its own absorption, but the underlying regulatory mechanisms are not known. We hypothesized that glucose transporter (GLUT) protein, member 5 (GLUT5) is the primary fructose transporter and that fructose absorption via GLUT5, metabolism via ketohexokinase (KHK), as well as GLUT5 trafficking to the apical membrane via the Ras-related protein-in-brain 11 (Rab11)a-dependent endosomes are each required for regulation. Introducing fructose but not lysine and glucose solutions into the lumen increased by 2- to 10-fold the heterogeneous nuclear RNA, mRNA, protein, and activity levels of GLUT5 in adult wild-type mice consuming chow. Levels of GLUT5 were >100-fold that of candidate apical fructose transporters GLUTs 7, 8, and 12 whose expression, and that of GLUT 2 and the sodium-dependent glucose transporter protein 1 (SGLT1), was not regulated by luminal fructose. GLUT5-knockout (KO) mice exhibited no facilitative fructose transport and no compensatory increases in activity and expression of SGLT1 and other GLUTs. Fructose could not up-regulate GLUT5 in GLUT5-KO, KHK-KO, and intestinal epithelial cellspecific Rab11a-KO mice. The fructose-specific metabolite glyceraldehyde did not increase GLUT5 expression. GLUT5 is the primary transporter responsible for facilitative absorption of fructose, and its regulation specifically requires fructose uptake and metabolism and normal GLUT5 trafficking to the apical membrane.—Patel, C., Douard, V., Yu, S., Gao, N., Ferraris, R. P. Transport, metabolism, and endosomal trafficking-dependent regulation of intestinal fructose absorption. FASEB J. 29, 000–000 (2015). www.fasebj.org ABSTRACT

Key Words: GLUT5 • ketohexokinase • Rab11a • sugar IN 2004, THE AVERAGE PER capita consumption of fructose in the United States was ;49 g/d, up 33% from ;37 g/d in 1978 (1), though a recent survey indicated a slight decrease beginning in 2008 (2). Excessive consumption of fructose has been tightly linked to the development of the metabolic syndrome of insulin resistance, dyslipidemia, Abbreviations: ARE, apical recycling endosome; Ef1a, elongation factor 1a; G6Pase, glucose-6-phosphatase; GLUT, glucose transporter; GLUT5, glucose transporter protein, member 5; hnRNA, heterogeneous nuclear RNA; HZ, heterozygous; IEC, intestinal epithelial cell; KHK, ketohexokinase; KO, knockout; Rab11, Ras-related protein-in-brain 11; SGLT1, sodium-dependent glucose transporter protein 1; WT, wild-type

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hypertension, and obesity (3). The small intestine via its transporters determines the amount of nutrients that enters into the blood and, thus, regulates the rate at which these nutrients become available to other organs. Fructose is absorbed into the cytosol by glucose transporter (GLUT) protein, member 5 (GLUT5) (Km, 6–14 mM; Slc2a5), a member of the facilitative GLUT family (4). GLUT5 is significantly expressed in epithelial cells of the small intestine, renal proximal tubule, as well as testis (4). Basolateral transport of fructose from the enterocyte to blood is mediated by GLUT2 (5). GLUT2 is thought to also be recruited to, and contribute to sugar transport across, the apical membrane (6). GLUTs 7, 8, and 12 are expressed in small intestinal cells (5), with GLUT8 regulating intestinal fructose transport (7). After entering into the cell, some fructose is phosphorylated by ketohexokinase (KHK or fructokinase) to fructose1-phosphate, which is further broken down to glyceraldehyde and dihydroxyacetone phosphate by aldolase-B. Fructolysis bypasses the major rate-limiting enzyme in glycolysis, phosphofructokinase. Without feedback inhibition, phosphorylation to fructose-1-phosphate rapidly depletes cellular ATP levels (8–10). KHK exists in 2 isoforms, KHK-A and KHK-C, but only the latter is considered the major fructosemetabolizing enzyme. KHK-C is mainly expressed in liver, intestine, and kidney and has a 10-fold higher affinity for fructose compared with KHK-A (11). Ras-related protein-in-brain 11 (Rab11) is an important guanosine 59-triphosphatase associated with recycling endosomes involved in endocytic and exocytic protein pathways (12) and in the trafficking of apical sugar hydrolases to the apical membrane (13). There are 3 isoforms, but only Rab11a is ubiquitously expressed in most tissues (14). Specifically, Rab11a is mainly associated with the apical recycling endosome (ARE) in polarized epithelia and regulates movement of apical proteins dipeptidylpeptidase 4 and alkaline phosphatase to the apical 1 Current affiliation: Unit´e Mixte de Recherche 1913Microbiologie de l’Alimentation au Service de la Sant´e, Institut National de la Recherche Agronomique, Domaine de Vilvert, Jouy-en-Josas, France. 2 Correspondence: Department of Pharmacology and Physiology, New Jersey Medical School, Rutgers University, 185 South Orange Ave., MSB H-621, Newark, NJ 07103, USA. E-mail: [email protected] doi: 10.1096/fj.15-272195 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

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membrane (15). The trafficking mechanism by which GLUT5 reaches the apical membrane and the role of Rab11a-mediated trafficking in fructose-induced regulation have not been investigated. GLUT5 plays a vital role in regulating the entry of fructose in our body. The wide array of adverse outcomes caused by excessive fructose consumption occurs only when fructose reaches vulnerable organ systems via the systemic circulation and perturbs their normal functions. Deletion of GLUT5 inhibits intestinal fructose absorption and reduces serum fructose concentration (16). Dietary fructose specifically upregulates GLUT5 mRNA and protein synthesis, leading to increased rates of fructose absorption (17). Although regulation of GLUT5 expression has been the subject of several studies [see review by (3)], the critical roles of transport, metabolism, and trafficking need to be determined. We used genetically modified mice to test the hypotheses that GLUT5 mediates fructose transport and that GLUT5mediated fructose transport, KHK-mediated fructolysis, and Rab11a-mediated GLUT5 trafficking are each required for fructose to induce expression of GLUT5. Because sweet taste receptors able to sense luminal sugars and artificial sweeteners have been localized in the small intestine (18), fructose may not need to enter the enterocytes for induction to occur. The role of fructose transport in GLUT5 regulation was evaluated using GLUT5-knockout (KO) mice, with the response of known fructose-responsive genes [glucose-6phosphatase (G6Pase); G6pc] used as readouts. Intracellular fructose by itself or its metabolites may induce expression; thus, the role of metabolism was tested using KHK-KO mice. Once GLUT5 expression and synthesis have been upregulated by dietary fructose, GLUT5 appears in abundance at the apical membrane, yet nothing is known about its trafficking. Thus, we used intestinal epithelial cell-specific Rab11a-KO (Rab11aDIEC) mice to determine whether

GLUT5 uses the Rab11a-dependent ARE mechanism to populate the apical membrane. MATERIALS AND METHODS Animals All the procedures conducted in this current study were approved by the Institutional Animal Care and Use Committee, New Jersey Medical School, Rutgers University. Tissues from wild-type (WT) and genetically modified mice [GLUT52/2 (KO), GLUT5+/2 (heterozygous; HZ), KHK2/2 (KO), KHK+/2 (HZ), and Rab11aDIEC] were used. Although GLUT5-KO and KHK-KO mice were lacking respective proteins globally, Rab11aDIEC mice were lacking Rab11a in IECs only because global Rab11a deletion is lethal in utero (12). All mice were in a temperature-controlled environment with a 12:12-h light-dark cycle. Mice had free access to water and a regular nonpurified diet (Purina Mills, Richmond, IN, USA). Same-age WT (C57BL/6 for KHK-KO and GLUT5-KO and 129/B6 for Rab11aDIEC) were used as a control in all the experiments involving GLUT5-KO, KHK-KO, or Rab11aDIEC mice. WT (C57BL/6) mice were descendants from HZ litters of initial breeding between founder WT and GLUT5-KO or KHK-KO mice. WT (129/B6) mice used for the Rab11aDIEC experiment were same-age littermates. Generation of GLUT5-KO (19), KHK-KO (20), and Rab11aDIEC (12) mice was described in detail previously. Genetic modifications in these mice have been validated using primer sequences in (12, 19, 20). Although GLUT5-KO and KHK-KO mice showed normal phenotypes if fed fructose-free diets, Rab11aDIEC mice exhibited runting and growth retardation. Because there was high mortality in postweaning Rab11aDIEC mice due to severe inflammation (12), Rab11aDIEC mice were used at 18 d of age. Experimental design In the first study utilizing a 3 3 3 factorial design, 4- to 5-wk-old WT, GLUT5-HZ, and GLUT5-KO mice were randomly divided

Figure 1. GLUT5 abundance relative to GLUTs 8 and 12. The relative mRNA abundance of GLUT5, GLUT8, and GLUT12 was measured in the jejunal mucosa of WT (dark-gray bars), GLUT5-HZ (HET; light gray), and GLUT5-KO (black) (A) as well as in WT, KHK-HZ, and KHK-KO (B) mice gavaged with 30% lysine, glucose, or fructose (2 ml/100 g, ;0.3 ml per mouse) twice a day for 2.5 d, to introduce these solutions into the gut lumen. At all other times, mice had access to standard rodent diet and grew normally. Ef1a was used as a reference gene. Data for each GLUT (indicated as 5, 8, and 12 in the horizontal axis) were normalized to the GLUT5 in WT mice gavaged with lysine (means 6 SE; n = 4–6). Bars with different letters are significantly different. There is no GLUT5 in GLUT5-KO mice in (A) as indicated by the absence of bars. Abundance of GLUT5 mRNA was much greater compared with that of GLUT8 and GLUT12, except in GLUT5-KO mice. Moreover, expression of GLUT5, but not GLUT8 and GLUT12, was inducible with fructose in all mice, except in those without GLUT5 (A) and KHK (B).

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into 3 groups and then gavaged, under light anesthesia, with 30% lysine (nonsugar control), glucose (sugar control), or fructose solution at a dose of 2 ml/100 g body weight (;0.3 ml per mouse) twice a day for 2.5 d (n = 6 per genotype and diet). After gavage feeding, mice were immediately returned to the cages where they resumed normal activity and ad libitum feeding of rodent nonpurified diet. Gavage feeding was used as a method to introduce solutions containing fructose and control (glucose and a nonsugar control, lysine) nutrients into the gut lumen and stimulate expression of fructose-responsive genes. Mice were euthanized 4 h after the last (fifth) gavage. The proximal small intestine was used for measurements of rates of glucose and fructose transport, and the next 6–8 cm was scraped and snapped frozen in liquid nitrogen for future analyses. The second study consisted of 2 experiments. In the first experiment, the same protocol as in the first study was followed, except that WT, KHK-HZ, and KHK-KO mice were compared. In the second experiment, 4- to 5-wk-old WT and then KHK-KO mice (n = 6 each group) were anesthetized then gavaged with 15% glucose, fructose, or glyceraldehyde solution at a dose of 2 ml/100 g as previously described. A concentration of 15% was selected as compromise because preliminary work found that 30% glyceraldehyde was not tolerated well, whereas 10% fructose solutions failed to up-regulate GLUT5. In the third study, Rab11aDIEC and WT (18-d-old) littermates were each randomly divided into 2 groups (n = 4 each group) and gavaged with either 30% glucose or fructose solutions as previously described (12).

reverse, and annealing temperature) as follows: GLUT5 hnRNAintron/intron, 59-TGTACGACACCCTACCCTACTG-39, 59GGTTCGAAAGATAATGAACGTGT-39, and 60°C; GLUT5 hnRNAintron/exon, 59-TCAAGAAAGACAAAGAAACCAGATG-39, 59-GTCATCGTCTTGCTTTGGT-39, and 56°C; SGLT1 hnRNA, 59-GGCTGACATCTCAGTCATCGTCAT-39, 59-GGGATATCTCCACTG TAAGCCCAT-39, and 58°C; b-actin hnRNA, 59-TTCTTTGCAGCTCCTTCGTTGCCG-39, 59-TCTACACGCTAGGCG TAAAGTTGG-39, and 58°C; G6Pase, 59-GGCTCACTTTCCCCATCAGG-39, 59-ATCCAAGTGCGAAACCAAACAG-39, and 54°C; GLUT7, 59CGAGTGCTGGTGGGAATC-39, 59-TTCTGGGGAGCCAGTTCTC-39, and 56°C; GLUT8, 59-TGTCACTGGCATCCTCCTG39, 59-GGCATGTAGCACATGAGCAGA-39, and 57°C; and GLUT12, 59-GGAGCTAGCAAAGGCGAAC-39, 59-TTGAAGCTGTGTTGGCACTAA-39, and 54°C. Western blot analysis Western blot analysis was performed using 70 mg protein extracted from intestinal mucosal scrapes, following methods described earlier (23). Intestinal proteins were separated using a precast 4–20% Tris/HCl gel (Bio-Rad), transferred to a membrane, and analyzed with primary antibodies against GLUT5 (1:300; EMD Millipore, Billerica, MA, USA) (donated by Dr. Chris Cheeseman, University of Alberta, Edmonton, AB, Canada). All membranes were stripped and reprobed with primary antibody against the housekeeping protein b-actin (1:1000; EMD Millipore).

Fructose uptake assay Fructose and glucose uptake rates in the small intestine were determined by the everted sleeve method described previously (21). Briefly, a 1 cm segment of jejunum was everted and mounted on a grooved steel rod and preincubated at 37°C for 5 minutes in Ringer solution. The sleeves were then incubated at 37°C in an oxygenated solution containing either D-[14C]glucose for 1 minute or D-[14C]fructose for 2 minutes. L-[3H]glucose was used to correct for adherent fluid and passive diffusion of glucose or fructose. Total mRNA extraction and reverse-transcription reaction Total RNA was extracted from the small intestinal mucosa using TRIzol reagent (Invitrogen/Life Technologies, Carlsbad, CA, USA). Purification of RNA was done using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The cDNA was generated using 5 mg RNA by RT-PCR using the iCycler Thermal Cycler (Bio-Rad, Hercules, CA, USA) with the SuperScript III Reverse Transcriptase kit (Invitrogen/Life Technologies).

Real-time PCR The cDNA obtained above was then analyzed by real-time PCR using Mx3000P (Stratagene, La Jolla, CA, USA) as described in our earlier work (22). The mRNA (exonic primer sequence) and heterogeneous nuclear RNA (hnRNA) (exonic and intronic primer sequences) were designed using the Roche primer design software (http://www.roche-applied-science.com) and were purchased from Integrated DNA Technologies (Coralville, IA, USA). Elongation factor 1a (Ef1a) and b-actin were used as housekeeping genes for mRNA and hnRNA, respectively. Previously published primer sequences for EF1a, GLUT2, GLUT5, and sodiumdependent glucose transporter protein 1 (SGLT1) are listed in reference (9). Primers for the following genes are (forward,

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Figure 2. GLUT5 abundance relative to GLUT7. The relative mRNA abundance of GLUT5 and GLUT7 was measured in jejunal mucosa of WT, GLUT5-HZ, and GLUT5-KO (A) and WT, KHK-HZ, and KHK-KO (B) mice gavaged with lysine, glucose, or fructose as previously described. Ef1a was used as a reference gene. Data for GLUT7 were normalized to those for GLUT5 in WT gavaged with lysine (means 6 SE; n = 4–6). Bars with different letters are significantly different. Expression of GLUT7 was much less compared with that of GLUT5 and was not inducible by fructose.

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Immunohistochemistry

Statistical analyses

Paraffinized tissue sections (;5 mm) were incubated with primary antibody (1:200 rabbit anti-rat GLUT5; EMD Millipore), goat anti-human villin (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and with the nuclear stain TO-PRO-3 (1:1000; Invitrogen/Life Technologies) in 13 PBS containing 2% donkey serum (Sigma-Aldrich, St. Louis, MO, USA), 2% bovine serum albumin (Sigma-Aldrich), and 0.1% Triton X-100 overnight at 4°C in a dark humidified chamber. These were washed in PBS, then fluorochrome-labeled secondary antibodies Alexa 546-conjugated donkey anti-rabbit IgG (1:500; Life Technologies) for GLUT5 and Alexa 488conjugated rabbit anti-goat IgG (1:500; Life Technologies) for villin were applied for 1 h at room temperature. Tissues were washed again in PBS, mounted with fluorescent mounting medium (Dako North America, Carpinteria, CA, USA), then examined with a laser-scanning confocal microscope (Nikon Eclipse Ti; Tokyo, Japan). All images compared were obtained with the same settings of the microscope.

Data are presented as means 6 SE. A 2-way ANOVA analyzed the effects of nutrient solution and genotype. Differences were considered significant at P # 0.05. If an initial 2-way ANOVA indicated a significant effect of nutrient solution and/or genotype, a 1-way ANOVA followed by a least significant difference test (StatView; Abacus Concepts, Berkeley, CA, USA) were used to determine differences among means.

RESULTS Body weight and food consumption Because our studies involved only an occasional isocaloric gavage feeding of fructose and other substrates over 2.5 d into mice that otherwise had ad libitum access to a commercial nonpurified diet, mice exhibited normal

Figure 3. The effect of dietary fructose on GLUT5 expression and function. The effect of fructose feeding and GLUT5 deletion on relative rates of facilitated fructose (A) and active glucose (B) uptake. Fructose and glucose uptake rates were measured in isolated everted jejunal sleeves obtained from WT, GLUT5-HZ, or GLUT5-KO mice gavaged with lysine, glucose, or fructose as previously described, to introduce these solutions into the gut lumen. Otherwise, mice had access to a nonpurified diet ad libitum. Levels of GLUT5 [(C), with a Western blot depicting corresponding GLUT5 levels) and of SGLT1 (D), G6Pase (E ), and GLUT2 (F ) mRNA. In all panels, data were normalized to those of WT mice gavaged with lysine. Results are means 6 SE (n = 4–6 each group). GLUT5 expression was analyzed by Western blot [n = 2; lower panel in (C ) shows a representative blot] with b-actin as reference. Bars with different letters are significantly different as analyzed by 1-way ANOVA. GLUT2 and SGLT1 expression and activity are normal, yet there is no fructose transport, in GLUT5-KO mice, regardless of diet. GLUT5 deletion prevents fructoseinduced up-regulation.

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growth and body weight for their age and had similar feeding rates. GLUT5- and KHK-KO mice tolerated gavage feeding of fructose. In the studies involving GLUT5 deletion, average daily consumption rate (overall mean, 5 g/d) of the nonpurified diet was similar across diets and genotype. Mean initial (20.3 g) and final (21.5 g) body weights were also similar across diets and genotype. In the KHK experiments, feeding rate was similar (5.2 g/d) for all experimental groups. Mean initial (20.2 g) and final (21.4 g) body weights were also the same for all experimental groups. In the third study, however, the targeted deletion of Rab11a from intestinal cells markedly affected growth. Thus, Rab11aDIEC mice were used at 18 d of age, at the late weaning stage, and initial (WT, 8.9; Rab11aDIEC, 4.9 g) and final (WT, 11.1; Rab11aDIEC, 6.6 g) weights were different. This result is expected because the phenotype of Rab11aDIEC mice is characterized by runting and slower growth compared with WT mice. Here, no feeding rate was measured because mice were returned to their dams after gavage feeding. GLUT5 is the primary fructose transporter in mouse small intestine

relative to GLUT5, was very low (P , 0.0001), did not vary with fructose (P = 0.6), and did not change with GLUT5 deletion (P = 0.5; Fig. 2A). Similar findings were obtained using KHK-KO mice (Fig. 2B). These results indicate that GLUT7 is not the primary transporter for fructose, is not regulated by fructose, and its expression does not vary with GLUT5 and KHK deletion.

Effect of fructose feeding and GLUT5 deletion on facilitative fructose uptake The effect of fructose feeding and GLUT5 deletion on fructose transport was highly significant (P = 0.04 and P , 0.0001, respectively, by 2-way ANOVA; Fig. 3A). The interaction between fructose feeding and genotype was also significant (P = 0.05), indicating that the fructose effect depended on genotype. Fructose transport in WT mice fed fructose was .2-fold compared with that in WT mice gavaged with lysine or glucose (P , 0.003). GLUT5-KO mice exhibited a mean transport rate not significantly different from zero. Fructose transport was similar between WT and GLUT5-HZ mice when gavaged with either lysine or

There was no facilitated fructose uptake into the mucosa of everted intestinal sleeves of GLUT5-KO mice (0.02 6 0.10 nmol/mg/m for fructose fed and 0.14 6 0.15 nmol/mg/m for glucose fed; n = 4 each group). In contrast, fructose transport rate in WT mice fed glucose was 1.2 6 0.2 nmol/mg/m, whereas those fed fructose was 2.6 6 0.3 nmol/mg/m (n = 5 each group). Relative expression of GLUTs 5, 8, and 12 The mRNA expression of putative fructose transporters GLUT8 and GLUT12 was several orders of magnitude lower than that of GLUT5 in the small intestine of WT and GLUT5-HZ mice primed (by gavage) with fructose, glucose, or lysine solutions but consuming the same nonpurified diet (P # 0.0002; Fig. 1A). GLUT8 and GLUT12 expression also remained low in GLUT5-KO mice, which expressed no GLUT5. Although fructose feeding increased by 3-fold the mRNA expression of GLUT5 in WT mice (P , 0.01), it had no effect on GLUT8 and GLUT12 mRNA expression (P $ 0.7 in all cases). Similar results were obtained in the small intestine of WT, KHK-HZ, and KHK-KO mice consuming the same nonpurified diet and gavage fed lysine, glucose, or fructose (Fig. 1B). All mice expressed significant levels of GLUT5, but GLUT8 and GLUT12 mRNA expression was again extremely low, not responsive to the luminal fructose signal (P $ 0.5 for both transporters), and exhibited no compensation for KHK deletion. In contrast, fructose feeding enhanced GLUT5 by .5-fold (P , 0.0001), except in KHKKO mice. Thus, GLUT8 and GLUT12 mRNA expression was extremely low, did not respond to a luminal fructose signal, and did not compensate for GLUT5 deletion. Relative expression of GLUT5 and GLUT7 GLUT7 can transport both glucose and fructose and is expressed in the small intestine (24). Its mRNA expression, METABOLIC CONTROL OF FRUCTOSE ABSORPTION

Figure 4. Effects of fructose feeding on hnRNA expression of GLUT5. Levels of GLUT5 hnRNAintron/intron (A), GLUT5 hnRNAintron/exon (B), and SGLT1 hnRNA (C) were determined by RT-PCR using b-actin as a reference gene. GLUT5 but not SGLT1 hnRNA increased with fructose gavage.

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glucose, suggesting that GLUT5-HZ mRNA and protein expression is sufficient for residual fructose transport. In sharp contrast, active glucose transport in the small intestine of WT, GLUT5-HZ, or GLUT5-KO mice was similar in all groups of mice and thus independent of diet (P = 0.6) and of genotype (P = 0.4, by 2-way ANOVA; Fig. 3B). Effect of fructose feeding and GLUT5 deletion on GLUT5 expression The effect of fructose feeding and GLUT5 deletion on mRNA expression of GLUT5 was highly significant (P = 0.0001 for both factors; Fig. 3C). The effect of fructose feeding on GLUT5 mRNA expression was modified by genotype (Pinteraction, 0.001). GLUT5 mRNA expression in WT mice fed fructose was 6-fold greater compared with those gavaged with lysine or glucose (P , 0.0001). Fructose-induced increases in GLUT5 were similar to those of mRNA levels. Interestingly, the mRNA expression of GLUT5 in GLUT5-HZ mice fed fructose was half of that observed in WT mice. As expected, no GLUT5 transcript was detected in GLUT5-KO mice. The effect of fructose feeding and GLUT5 deletion on mRNA expression of the major GLUT, SGLT1, and of the basolateral transporter, GLUT2, was not significant, suggesting that GLUT5 deletion was not compensated by changes in SGLT1 and GLUT2 mRNA expression (Fig. 3D, F). The effect of

fructose on GLUT5 mRNA expression correlated with fructose transport in WT, GLUT5-HZ, and GLUT5-KO mice. The effect of fructose feeding (P # 0.03) and GLUT5 deletion (P # 0.007) on the mRNA expression of G6Pase was highly significant (Fig. 3E). In WT mice, fructose feeding increased the mRNA expression of G6Pase by $3fold (P # 0.01) relative to those fed with lysine or glucose. This increase in mRNA expression of G6Pase observed in WT mice was clearly prevented in GLUT5-KO mice gavaged with fructose. Deletion of GLUT5 did not affect the basal mRNA expression of G6Pase because there was no significant difference in the levels of these enzymes in GLUT5-KO mice fed lysine, glucose, or fructose (P $ 0.6). The finding that GLUT5 deletion abolishes the ability of dietary fructose to regulate a known fructose-responsive gene suggests that fructose transport is likely required for fructose-induced regulation of these genes. To assess the effect of fructose on transcriptional activation of GLUT5, various primers were designed that could recognize GLUT5 hnRNA. Primer sets were designed from sequences either within intron 4 only or between intron 13 and exon 14 of Slc2a5. The effect of fructose feeding and GLUT5 deletion on levels of GLUT5 hnRNAintron/intron (P = 0.002 and P , 0.0001, respectively; Fig. 4A) and of GLUT5 hnRNAintron/exon (P = 0.01 and P , 0.0001, respectively; Fig. 4B) was significant. Interaction between fructose feeding and genotype was also significant for both GLUT5 hnRNAintron/intron and GLUT5 hnRNAintron/exon

Figure 5. Effect of KHK deletion on regulation of GLUT5 by fructose. The effect of fructose feeding and KHK deletion on facilitated fructose (A) and active glucose uptake (B) rates in WT, KHK-HZ, or KHK-KO mice gavaged with lysine, glucose, or fructose. Results are means 6 SE (n = 4–6). Expression of GLUT5 mRNA and protein (C ) and of SGLT1 mRNA (D). GLUT5 expression was analyzed by Western blot [n = 2; panel in (C ) shows a representative blot] with b-actin as a reference gene. KHKmediated metabolism is required for fructose-induced up-regulation of GLUT5.

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(P = 0.001 for both cases). In WT mice, fructose gavage increased GLUT5 hnRNAintron/intron and GLUT5 hnRNAintron/exon expression by 3- to 5-fold compared with those gavaged with lysine or glucose. The changes in GLUT5 hnRNA expression are tightly correlated with those of GLUT5 mRNA expression and suggest transcriptional activation. The effect of fructose on GLUT5 hnRNA was specific because SGLT1 hnRNA expression was not affected by fructose feeding or GLUT5 deletion (Fig. 4C). The effect of KHK deletion on fructose-induced fructose transport By 2-way ANOVA, the effects of fructose feeding and KHK deletion on fructose transport were highly significant (P , 0.0001 and P = 0.003, respectively). The interaction between genotype and fructose feeding was also significant (P = 0.01), suggesting that the effect of fructose feeding on fructose uptake depended upon KHK. Fructose transport in WT mice fed fructose was 2.5-fold greater compared with that in mice gavaged with lysine or glucose (P , 0.0001). This increase in fructose transport was clearly prevented in KHK-KO mice whose fructose uptake was similar (P . 0.3) to WT fed lysine or glucose (Fig. 5A). Interestingly, the response of KHK-HZ mice was similar to that of WT mice fed fructose. Fructose transport in WT and KHK-HZ mice fed lysine or glucose was similar to that in the KHKKO mice (P $ 0.6 in all cases). This suggests that deletion of KHK did not affect residual fructose transport. In sharp contrast, active glucose transport in the small intestine of

WT, KHK-HZ, or KHK-KO mice was similar in all groups of mice and thus independent of diet and of genotype (Fig. 5B). The effect of fructose feeding and KHK deletion on GLUT5 expression The effect of fructose feeding and KHK deletion on mRNA expression of GLUT5 was highly significant (P = 0.0002 and P = 0.02, respectively; Fig. 5C). The effect of fructose was dependent on genotype (Pinteraction, 0.01). GLUT5 mRNA expression in WT and KHK-HZ mice fed fructose was 5- to 8-fold greater compared with those gavaged with lysine or glucose (P = 0.001 and P = 0.0007, respectively). GLUT5 expression was correlated with GLUT5 mRNA expression and with fructose uptake. This increase in GLUT5 and mRNA expression in WT mice was clearly prevented in KHK-KO mice gavaged with fructose. There was no significant difference in GLUT5 mRNA expression among WT, KHK-HZ, and KHK-KO mice gavaged with either lysine or glucose (P $ 0.6 in all cases), suggesting that these levels constitute the baseline expression. SGLT1 expression in WT, KHK-HZ, or KHK-KO mice did not vary with diet and KHK deletion (Fig. 5D). Effect of glyceraldehyde on sugar transport and gene expression Because GLUT5 up-regulation is KHK dependent, here, we determined whether a fructose-specific metabolite, glyceraldehyde, can up-regulate GLUT5.

Figure 6. Effect of glyceraldehyde on fructose uptake in WT mice. The effect of glyceraldehyde feeding on facilitated fructose (A) and active glucose (B) uptake determined in everted jejunal sleeves of WT mice gavaged with 15% glucose, fructose, or glyceraldehyde. The expression of GLUT5 mRNA [(C ), with a Western blot depicting corresponding protein levels] and SGLT1 mRNA (D) was also determined. In all panels, data were normalized to WT mice gavaged with glucose. Glyceraldehyde seems to increase fructose uptake by nongenomic mechanisms.

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WT mice Fructose uptake rate increased by 2-fold in mice gavaged with fructose and with glyceraldehyde (P , 0.0003, by 1-way ANOVA) compared with that in mice fed glucose (Fig. 6A). There was no effect on glucose uptake (P = 0.5; Fig. 6B). Fructose, but not glyceraldehyde, feeding increased GLUT5 mRNA expression (P , 0.0001; Fig. 6C). GLUT5 expression displayed a pattern similar to that of GLUT5 mRNA. Surprisingly, SGLT1 expression decreased significantly with glyceraldehyde feeding (P = 0.04; Fig. 6D). GLUT2 mRNA expression was similar among mice fed fructose, glucose, and glyceraldehyde (data not shown). These results indicate that paradoxically, glyceraldehyde can increase fructose uptake without increasing GLUT5 mRNA and protein expression.

KHK-KO mice We repeated the glyceraldehyde feeding experiment in KHK-KO mice that was expected to have less endogenous glyceraldehyde. Interestingly, glyceraldehyde significantly increased the rate of fructose uptake in KHK-KO mice (P = 0.001; Fig. 7A), an effect similar to what we previously observed in WT mice (Fig. 6A). Fructose feeding did not increase fructose uptake in KHK-KO mice, reconfirming previous observations. Glucose transport in the small intestine of KHK-KO mice was independent (P = 0.2; Fig. 7B)

of nutrient solution. The mRNA and protein expression of GLUT5 was similar among KHK-KO mice gavaged with glucose, fructose, and glyceraldehyde (P = 0.2; Fig. 7C). Like the results in WT mice, glyceraldehyde decreased the mRNA expression of SGLT1 (P = 0.03; Fig. 7D). GLUT2 mRNA expression was similar among mice fed fructose, glucose, and glyceraldehyde (data not shown). Rab11a-mediated trafficking is involved in fructoseinduced GLUT5 regulation The effect of fructose feeding and Rab11a deletion on fructose uptake The effect of fructose feeding and Rab11a deletion on fructose uptake was highly significant (P = 0.0003 and P , 0.0001, respectively; Fig. 8A). The interaction between fructose feeding and genotype was also significant (P = 0.04), suggesting that the effect of fructose on fructose uptake depended upon the genotype. The fructoseinduced increase in fructose transport in WT mice (P , 0.0001) was blunted in Rab11aDIEC mice, implying an important role of Rab11a-mediated protein trafficking in regulating fructose uptake. However, deletion of Rab11a also decreased glucose uptake (P = 0.003; Fig. 8B), regardless of diet. Thus, there are 2 effects of Rab11a deletion: reduction in the number of constitutive functional transporters in the apical membrane, and prevention of fructose-induced regulation of GLUT5.

Figure 7. Effect of glyceraldehyde on fructose uptake in KHK-KO mice. The effect of glyceraldehyde feeding on facilitated fructose (A) and active glucose (B) uptake. The effect of glyceraldehyde was also determined on the mRNA expression of GLUT5 [(C), with a Western blot depicting corresponding protein levels] and of SGLT1 (D). Glyceraldehyde increases fructose uptake but does not induce GLUT5 mRNA expression in KHK-KO mice.

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Figure 8. The effect of Rab11a deletion on GLUT5 regulation by fructose. The effect of fructose feeding and Rab11a deletion on facilitated fructose (A) and active glucose (B) uptake and on the expression of GLUT5 mRNA (C) and SGLT1 mRNA (D) was determined as previously described. Briefly, uptake was measured using everted sleeves from the jejunum of 18-d-old WT and Rab11aDIEC mice gavaged with 30% glucose or fructose (2 ml/100 g) twice a day for 2.5 consecutive days. Results are means 6 SE (n = 4–6). Both baseline fructose uptake in glucose-fed mice and fructose-induced increases in fructose uptake seem inhibited in Rab11aDIEC mice.

The effect of fructose feeding and Rab11a deletion on GLUT5 expression The effect of fructose feeding and Rab11a deletion on GLUT5 mRNA expression was highly significant (P = 0.04 and P = 0.003, respectively, by 2-way ANOVA; Fig. 8C). Fructose feeding increased GLUT5 mRNA expression in WT mice by 8-fold (P , 0.003), but this increase was prevented in Rab11aDIEC mice (P . 0.2). Rab11a deletion also reduced by ;50% levels of SGLT1 mRNA in mice fed glucose or fructose (Fig. 8D). The mRNA expression of GLUT5 and SGLT1 correlated with fructose and glucose uptake results.

also indicated in the merged panels of Rab11aDIEC mice gavaged with either glucose or fructose (Fig. 9I, L), where GLUT5 failed to colocalize with villin. Differences in colocalization of GLUT5 with villin were more remarkable when intestinal sections of WT and of Rab11aDIEC mice gavaged with fructose are compared (arrows in Fig. 9C, I as well as F, L). These results are in accordance with similar patterns of fructose uptake and GLUT5 mRNA expression. GLUT5 and villin antibodies were specific as indicated by the absence of immunofluorescence in WT and Rab11aDIEC sections using secondary antibodies only (not shown). DISCUSSION

Effect of Rab11a deletion on GLUT5 trafficking to the apical membrane Because removal of Rab11a prevented fructose-induced increases not only in the expression of fructose-responsive genes in the cytosol but also in fructose uptake rate across the apical membrane, we chronicled its effects on GLUT5 levels in the apical membrane. Fructose but not glucose feeding increased GLUT5 immunofluorescence in the cytosol and along the apical membrane of enterocytes of WT mice (compare white arrows in Fig. 9A, D). GLUT5 was colocalized with the intestinal apical membrane marker villin as indicated by the brighter color of the apical membrane in the merged panels (compare yellow arrows in Fig. 9C, F). Fructose feeding also increased GLUT5 immunofluorescence in the cytosolic but not apical membrane regions of Rab11aDIEC enterocytes (compare Fig. 9G and J). The absence of GLUT5 in the apical membrane is METABOLIC CONTROL OF FRUCTOSE ABSORPTION

The 2 novel findings in this study are that fructoseinduced increases in GLUT5 hnRNA, mRNA, and protein expression as well as in GLUT5 activity are dependent on GLUT5-mediated transport and KHKmediated metabolism and that the targeted deletion of Rab11a recycling endosome in intestinal epithelia partly interrupts GLUT5 trafficking to the apical membrane, reducing basal fructose transport as well as preventing fructose-induced increases in GLUT5 expression. The other related, highly interesting findings are that 1) GLUT5, not GLUTs 7, 8, and 12, is the major and primary intestinal fructose transporter; 2) GLUT7, GLUT8, and GLUT12, whereas expressed in the intestine at low levels, do not respond to luminal sugars and to removal of GLUT5 and KHK; 3) the fructosespecific metabolite glyceraldehyde modulates GLUT5 function independent of transcriptional and translational 9

Figure 9. Effect of Rab11a-mediated trafficking on GLUT5 levels in the apical membrane. In WT mice fed glucose (WT-G), GLUT5 was expressed at low abundance in the enterocyte cytosol (A) (please see inset) and in the apical membrane (white arrow) where it colocalized with the membrane biomarker villin (B, orange arrow) as shown in the merged panel (C, yellow arrow). When WT mice were fed fructose (WTF), GLUT5 levels seemed to increase markedly in the cytosol and apical membrane (D–F ). GLUT5 was also expressed in moderate amounts in the Rab11aDIEC mice fed glucose (G–I ). When Rab11aDIEC mice were fed fructose, GLUT5 levels seemed to increase in the cytosol (J, compare with A and G) but not in the apical membrane where the merged panel reflects mainly green immunofluorescence from villin (K and L). Thus, the absence of Rab11a from IECs prevents most GLUT5 from being inserted in the apical membrane (compare D to J and F to L), reducing rates of fructose uptake. White scale bar, 20 mm.

mechanisms; and 4) Rab11a is also involved in SGLT1 trafficking to the apical membrane of the small intestine. GLUT5 is an essential intestinal fructose transporter The absence of any significant L-glucose-corrected, carriermediated fructose uptake in vitro in GLUT5-KO mice strongly suggests that GLUT5 is the major apical transporter for fructose and may be responsible for virtually all transcellular fructose uptake from the lumen. This finding expands and extends the findings of Barone et al. (16), who determined that total fructose uptake into apical membrane vesicles from WT to be 4-fold greater than that into vesicles of GLUT5-KO mice. The residual uptake in their GLUT5-KO mice (which should have no carrier-mediated uptake) may be due to fructose in the extravesicular space (adsorbed fructose) and to diffusive fructose transport. Because L-glucose is not recognized by eukaryotic enzymes as well as transporters and because its diffusive properties are similar to fructose, we corrected for adsorption and diffusion by subtracting 3H-Lglucose “uptake” from total 14C-D-fructose uptake and found insignificant net (carrier-mediated) fructose uptake in GLUT5-KO mice. GLUT2 and SGLT1 expression as well as activity were normal, yet there was no fructose transport in GLUT5-KO mice, regardless of diet. Coupled with 10

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observations that GLUT5-KO mice exhibit watery diarrhea if fed fructose-containing foods (16), these findings fail to support suggestions that GLUT5 is responsible for only 40% of the fructose uptake under a high-fructose diet, with the majority of transport activity mediated by GLUT2 proposed to traffic rapidly and transiently to the apical membrane (6). The hypothesized transient translocation of GLUT2, classically considered to be the basolateral sugar transporter, to the apical membrane (6) is not supported by data from the GLUT5-KO mice, from humans without SGLT1 that suffer from glucose galactose malabsorption (25), and from SGLT1-KO mice that cannot absorb glucose (26). Using Caco2 cells, GLUT12 has been shown to transport fructose and to respond to high fructose (27), but our results using intact tissue do not fully support these findings. GLUT8 was hypothesized to regulate enterocyte fructose transport by regulating GLUT12 in vivo (7), but this hypothesis was not supported by our findings in mice. The insulin-sensitive GLUT12 actually selectively transports glucose and is found in many tissues where it may be overexpressed in cancer to enhance glycolytic metabolism (28). GLUT7 is a hexose transporter that also transports fructose and is closely related structurally to GLUT5 (5). Unlike GLUT5, however, it is expressed and localized in the apical membrane of both the small intestine and colon

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Figure 10. A proposed model of GLUT5 regulation by its substrate fructose. First, fructose (F) is required to cross F F F the apical membrane via M M F GLUT5 (step 1). Once in the F F GL M cytosol, fructose needs to be metabolized by KHK (2) to an unidentified metabolite (M) for regulation to take place. F F F The signal for Slc2a5 up-regulation seems linked to this step F F F and is shared by other fructoseresponsive genes like G6Pc. The fructose-specific metabolite glyR ceraldehyde (GL) does not inF G F crease GLUT5 expression and G thus is not the regulatory M. (3). F Na G G Our current and previous studG ies suggest that once metabolism Na G occurs, GLUT5 transcription increases, leading to increased GLUT5 mRNA and then protein (4). A Rab11a (R)-dependent endosome is likely involved in the trafficking of newly synthesized GLUT5 from the endoplasmic reticulum (ER) and Golgi complex to the apical membrane because fructose-induced increases in GLUT5 abundance and activity in the apical membrane are prevented in Rab11aDIEC mice (5). SGLT1 absorbs glucose (G), and both glucose and fructose exit the cell by GLUT2.

(24). The true substrate of GLUT7 is still not known, but it has a high affinity for fructose (Km, 0.5 mM). GLUT7 expression levels vary with dietary carbohydrate levels (24), and not with luminal fructose levels, indicating that its substrate is likely not fructose. Fructose-induced GLUT5 up-regulation involves transcriptional activation We and others used the transcription inhibitor actinomycin D in combination with the translation inhibitor cyclohexamide to prove that fructose-induced GLUT5 upregulation involves de novo mRNA and protein synthesis (17, 29). In fact, fructose regulation increases polymerase II binding and histone H3 acetylation to the GLUT5 promoter (30). Fructose feeding also induced acetylation of H3 and H4 proteins in Slc2a5 and in other fructose-inducible genes (31). These early studies and our current findings on GLUT5 hnRNA confirm the involvement of transcriptional components in fructose-induced increases in rates of intestinal fructose transport. In general, many transporters have been shown to be regulated at the level of transcription, notably many members of the large family of ABC drug transporters (32). In fact, the level of hnRNA, the initial product of gene transcription, has now been increasingly used as a reliable marker of transcriptional activation (33). Fructose metabolism is required for GLUT5 induction The nonmetabolizable fructose analog 3-O-methylfructose could not induce GLUT5 activity and modestly METABOLIC CONTROL OF FRUCTOSE ABSORPTION

enhanced mRNA expression (17). However, because of the much lower affinity of GLUT5 to 3-O-methylfructose compared with fructose, inhibition of GLUT5 induction may be due to poor absorption of this analog. The KHK-KO mice provided unequivocal proof that fructose metabolism is required and that the signal must be specific for GLUT5 because GLUT2 and SGLT1 expression was not affected. Similar links between metabolism and transport have been demonstrated because regulation of hepatic GLUT2 and asparagine synthetase expression by glucose depends on glucose metabolism (34, 35). Because fructose metabolism is its primary source, endogenous glyceraldehyde is a likely candidate as the signal specifically regulating fructose-responsive genes. Dietary glyceraldehyde can be transported into intestinal cells via a saturable, competitively inhibited, electrogenic, and Na+-dependent glyceraldehyde transporter known to be expressed in the pancreas (36). Up-regulation by glyceraldehyde of fructose transport is clearly nongenomic in nature in WT mice. Rab11a-mediated endosomal trafficking is required for GLUT5 regulation Rab8a, Rab11, and Rab13 mediate the trafficking of GLUT protein, member 4 to the surface membrane of skeletal muscle in an insulin-dependent manner (37). These Rabs also regulate the sorting of membrane proteins in polarized epithelia. Thus, Rab8 mediates the trafficking of the proton-dependent peptide transporter PEPT1 and of SGLT1 from the intestinal cytosol to the apical membrane in vivo (38). An isozyme of 11

Rab11 targets the enzymes phlorizin hydrolase and sucrose-isomaltase to the apical membrane of MadinDarby canine kidney cells (13). The targeted removal of Rab11a from intestinal epithelia resulted in a dilated lumen, shortened villi, and reduced number of goblet cells (12), as well as in the inappropriate accumulation of known apical proteins in the cytosol (15). Because Rab11a deletion reduces the baseline activity and mRNA expression of GLUT5, SGLT1 (Fig. 8), and GLUT2 (data not shown), faulty trafficking of GLUT5 and SGLT1 to the apical membrane could result in reduced amounts of sugars entering the cell, reducing the signals maintaining steady-state expression of genes involved in sugar transport. Rab11a deletion also stops newly synthesized GLUT5 from reaching the apical membrane, preventing the rapid influx of additional dietary fructose and inhibiting up-regulation as depicted in our model (Fig. 10). The authors are grateful to Drs. E. David, I. Monteiro, P. Tharabenjasin, and E. Topaktas [New Jersey Medical School (NJMS)] as well as to Ms. J. Lee (NJMS), C. Monteiro (NJMS), D. O’Neill (University of Alberta), and Mr. D. Simon (NJMS) for help with experiments and/or for valuable discussion. The authors are especially grateful to Profs. C. Cheeseman (University of Alberta), R. Johnson (University of Colorado), and J. Zuo (St. Jude’s Children’s Hospital) for the gifts of GLUT5 antibody, the KHK-KO, and the GLUT5-KO mice, respectively. C.P. maintained animal models, conducted all experiments, analyzed the data, and helped in experimental design and manuscript preparation. V.D. helped design the studies, perform experiments, analyze data, and finalize the manuscript. S.Y. helped maintain animals, perform experiments, and analyze data. N.G. analyzed data as well as revised and finalized the manuscript. R.P.F. helped design the experiment, coordinated activities of coauthors and collaborators, helped analyze all data, then revised as well as finalized the manuscript. This work was supported by U.S. National Science Foundation Grants IOS-1121049 and 1456673 (to R.P.F.). The study also received support with U.S. National Institutes of Health Grants DK085194, DK093809, DK102934 from the National Institute of Diabetes and Digestive and Kidney Diseases, and CA178599 from the National Cancer Institute (to N.G.). This work was performed in partial fulfillment of the Ph.D. degree in biomedical sciences at the Graduate School of Biomedical Sciences, Rutgers University for C.P. The authors declare no conflicts of interest.

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