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Transport, metabolism, and endosomal trafficking-dependent regulation of intestinal fructose absorption - PubMed

Transport, metabolism, and endosomal trafficking-dependent regulation of intestinal fructose absorption

Chirag Patel et al. FASEB J. 2015 Sep.

Abstract

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 cell-specific 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.

Keywords: GLUT5; Rab11a; ketohexokinase; sugar.

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Figures

Figure 1.
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. Ef1α 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 ±

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).

Figure 2.
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. Ef1α was used as a reference gene. Data for GLUT7 were normalized to those for GLUT5 in WT gavaged with lysine (means ±

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.

Figure 3.
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 ±

se

(n = 4–6 each group). GLUT5 expression was analyzed by Western blot [n = 2; lower panel in (C) shows a representative blot] with β-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 fructose-induced up-regulation.

Figure 4.
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 β-actin as a reference gene. GLUT5 but not SGLT1 hnRNA increased with fructose gavage.

Figure 5.
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 ±

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 β-actin as a reference gene. KHK-mediated metabolism is required for fructose-induced up-regulation of GLUT5.

Figure 6.
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.

Figure 7.
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.

Figure 8.
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 Rab11aΔIEC mice gavaged with 30% glucose or fructose (2 ml/100 g) twice a day for 2.5 consecutive days. Results are means ±

se

(n = 4–6). Both baseline fructose uptake in glucose-fed mice and fructose-induced increases in fructose uptake seem inhibited in Rab11aΔIEC mice.

Figure 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 (WT-F), GLUT5 levels seemed to increase markedly in the cytosol and apical membrane (D–F). GLUT5 was also expressed in moderate amounts in the Rab11aΔIEC mice fed glucose (G–I). When Rab11aΔIEC 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 µm.

Figure 10.
Figure 10.

A proposed model of GLUT5 regulation by its substrate fructose. First, fructose (F) is required to cross the apical membrane via GLUT5 (step 1). Once in the cytosol, fructose needs to be metabolized by KHK (2) to an unidentified metabolite (M) for regulation to take place. The signal for Slc2a5 up-regulation seems linked to this step and is shared by other fructose-responsive genes like G6Pc. The fructose-specific metabolite glyceraldehyde (GL) does not increase GLUT5 expression and thus is not the regulatory M. (3). Our current and previous studies suggest that once metabolism 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 Rab11aΔIEC mice (5). SGLT1 absorbs glucose (G), and both glucose and fructose exit the cell by GLUT2.

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