Veronique Douard

Regulation of the fructose transporter GLUT5 in health and disease

Fructose is now such an important component of human diets that increasing attention is being focused on the fructose transporter GLUT5. In this review, we describe the regulation of GLUT5 not only in the intestine and testis, where it was first discovered, but also in the kidney, skeletal muscle, fat tissue, and brain where increasing numbers of cell types have been found to have GLUT5. GLUT5 expression levels and fructose uptake rates are also significantly affected by diabetes, hypertension, obesity, and inflammation and seem to be induced during carcinogenesis, particularly in the mammary glands. We end by highlighting research areas that should yield information needed to better understand the role of GLUT5 during normal development, metabolic disturbances, and cancer.

Cdc42 and Rab8a are critical for intestinal stem cell division, survival, and differentiation in mice

The constant self renewal and differentiation of adult intestinal stem cells maintains a functional intestinal mucosa for a lifetime. However, the molecular mechanisms that regulate intestinal stem cell division and epithelial homeostasis are largely undefined. We report here that the small GTPases Cdc42 and Rab8a are critical regulators of these processes in mice. Conditional ablation of Cdc42 in the mouse intestinal epithelium resulted in the formation of large intracellular vacuolar structures containing microvilli (microvillus inclusion bodies) in epithelial enterocytes, a phenotype reminiscent of human microvillus inclusion disease (MVID), a devastating congenital intestinal disorder that results in severe nutrient deprivation. Further analysis revealed that Cdc42-deficient stem cells had cell division defects, reduced capacity for clonal expansion and differentiation into Paneth cells, and increased apoptosis. Cdc42 deficiency impaired Rab8a activation and its association with multiple effectors, and prevented trafficking of Rab8a vesicles to the midbody. This impeded cytokinesis, triggering crypt apoptosis and disrupting epithelial morphogenesis. Rab8a was also required for Cdc42-GTP activity in the intestinal epithelium, where continued cell division takes place. Furthermore, mice haploinsufficient for both Cdc42 and Rab8a in the intestine demonstrated abnormal crypt morphogenesis and epithelial transporter physiology, further supporting their functional interaction. These data suggest that defects of the stem cell niche can cause MVID. This hypothesis represents a conceptual departure from the conventional view of this disease, which has focused on the affected enterocytes, and suggests stem cell-based approaches could be beneficial to infants with this often lethal condition.

The role of fructose transporters in diseases linked to excessive fructose intake

Fructose intake has increased dramatically since humans were hunter‐gatherers, probably outpacing the capacity of human evolution to make physiologically healthy adaptations. Epidemiological data indicate that this increasing trend continued until recently. Excessive intakes that chronically increase portal and peripheral blood fructose concentrations to >1 and 0.1 mm, respectively, are now associated with numerous diseases and syndromes. The role of the fructose transporters GLUT5 and GLUT2 in causing, contributing to or exacerbating these diseases is not well known. GLUT5 expression seems extremely low in neonatal intestines, and limited absorptive capacities for fructose may explain the high incidence of malabsorption in infants and cause problems in adults unable to upregulate GLUT5 levels to match fructose concentrations in the diet. GLUT5‐ and GLUT2‐mediated fructose effects on intestinal electrolyte transporters, hepatic uric acid metabolism, as well as renal and cardiomyocyte function, may play a role in fructose‐induced hypertension. Likewise, GLUT2 may contribute to the development of non‐alcoholic fatty liver disease by facilitating the uptake of fructose. Finally, GLUT5 may play a role in the atypical growth of certain cancers and fat tissues. We also highlight research areas that should yield information needed to better understand the role of these GLUTs in fructose‐induced diseases.

Linseed Oil in the Maternal Diet during Gestation and Lactation Modifies Fatty Acid Composition, Mucosal Architecture, and Mast Cell Regulation of the Ileal Barrier in Piglets

In this study, we investigated the effect of supplementation of the maternal diet with linseed oil [rich in 18:3(n-3)] on fatty acid composition, mucosal architecture, and mast cell regulation of barrier function in piglet ileum. Sixteen sows were fed a lard (LAR)- or a linseed oil (LSO)-based diet during gestation and lactation. Fatty acid composition of maternal RBC at parturition and of milk at d 14 of lactation were determined. Fatty acid composition, villous-crypt structure, and permeability to horseradish peroxidase in Ussing chambers after mast cell degranulation were determined in the ileum of piglets at d 0, 7, and 28. At d 0, 18:3(n-3) and 20:5(n-3) levels were higher, but 22:6(n-3) and 20:4(n-6) levels were lower in both maternal RBC and piglet ileum of the LSO group. Levels of 18:3(n-3) were also higher in the milk of LSO sows. Levels of 18:3(n-3) were higher in LSO piglet ileum at d 7 and 28. Moreover, at d 28, 20:4(n-6) ileal levels tended (P = 0.09) to be lower in LSO than in LAR piglets, in parallel with a lower mRNA expression of Delta5 desaturase. LSO piglets had shorter villi at d 0 and shorter crypts at d 7 compared with LAR piglets. The effect of mast cell degranulation on ileal permeability decreased with age in both groups but reached a minimum sooner in the LSO group (d 7) than in the LAR group (d 28). In conclusion, linseed oil supplementation of the maternal diet profoundly modifies the fatty acid composition, structure, and physiology of the offspring ileum.

Diet-induced epigenetic regulation in vivo of the intestinal fructose transporter Glut5 during development of rat small intestine.

Metabolic complications arising from excessive fructose consumption are increasing dramatically even in young children, but little is known about ontogenetic mechanisms regulating Glut5 [glucose transporter 5; encoded by the Slc2a5 (solute carrier family 2 member 5) gene]. Glut5 expression is low postnatally and does not increase, unless luminal fructose and systemic glucocorticoids are present, until ≥ 14 days of age, suggesting substrate-inducible age- and hormone-sensitive regulation. In the present study, we perfused intestines of 10- and 20-day-old rats with either fructose or glucose then analysed the binding of Pol II (RNA polymerase II) and GR (glucocorticoid receptor), as well as acetylation of histones H3 and H4 by chromatin immunoprecipitation. Abundance of Glut5 mRNA increased only with fructose perfusion and age, a pattern that matched that of Pol II binding and histone H3 acetylation to the Glut5 promoter. Although many regions of the Glut5 promoter respond to developmental signals, fewer regions perceive dietary signals. Age- but not fructose-dependent expression of Sglt1 [sodium-dependent glucose co-transporter 1 encoded by the Slc5a1(solute carrier family 5 member 1) gene] also correlated with Pol II binding and histone H3 acetylation. In contrast, G6Pase (glucose-6-phosphatase; encoded by the G6pc gene) expression, which decreases with age and increases with fructose, is associated only with age-dependent changes in histone H4 acetylation. Induction of Glut5 during ontogenetic development appears to be specifically mediated by GR translocation to the nucleus and subsequent binding to the Glut5 promoter, whereas the glucocorticoid-independent regulation of Sglt1 by age was not associated with any GR binding to the Sglt1 promoter.

TLR sorting by Rab11 endosomes maintains intestinal epithelial‐microbial homeostasis

Compartmentalization of Toll‐like receptors (TLRs) in intestinal epithelial cells (IECs) regulates distinct immune responses to microbes; however, the specific cellular machinery that controls this mechanism has not been fully identified. Here we provide genetic evidences that the recycling endosomal compartment in enterocytes maintains a homeostatic TLR9 intracellular distribution, supporting mucosal tolerance to normal microbiota. Genetic ablation of a recycling endosome resident small GTPase, Rab11a, a gene adjacent to a Crohns disease risk locus, in mouse IECs and in Drosophila midgut caused epithelial cell‐intrinsic cytokine production, inflammatory bowel phenotype, and early mortality. Unlike wild‐type controls, germ‐free Rab11a‐deficient mouse intestines failed to tolerate the intraluminal stimulation of microbial agonists. Thus, Rab11a endosome controls intestinal host‐microbial homeostasis at least partially via sorting TLRs.

Dietary Fructose Inhibits Intestinal Calcium Absorption and Induces Vitamin D Insufficiency in CKD

Renal disease leads to perturbations in calcium and phosphate homeostasis and vitamin D metabolism. Dietary fructose aggravates chronic kidney disease (CKD), but whether it also worsens CKD-induced derangements in calcium and phosphate homeostasis is unknown. Here, we fed rats diets containing 60% glucose or fructose for 1 mo beginning 6 wk after 5/6 nephrectomy or sham operation. Nephrectomized rats had markedly greater kidney weight, blood urea nitrogen, and serum levels of creatinine, phosphate, and calcium-phosphate product; dietary fructose significantly exacerbated all of these outcomes. Expression and activity of intestinal phosphate transporter, which did not change after nephrectomy or dietary fructose, did not correlate with hyperphosphatemia in 5/6-nephrectomized rats. Intestinal transport of calcium, however, decreased with dietary fructose, probably because of fructose-mediated downregulation of calbindin 9k. Serum calcium levels, however, were unaffected by nephrectomy and diet. Finally, only 5/6-nephrectomized rats that received dietary fructose demonstrated marked reductions in 25-hydroxyvitamin D(3) and 1,25-dihydroxyvitamin D(3) levels, despite upregulation of 1alpha-hydroxylase. In summary, excess dietary fructose inhibits intestinal calcium absorption, induces marked vitamin D insufficiency in CKD, and exacerbates other classical symptoms of the disease. Future studies should evaluate the relevance of monitoring fructose consumption in patients with CKD.

Developmental reprogramming of rat GLUT5 requires glucocorticoid receptor translocation to the nucleus

Fructose consumption has increased dramatically but little is known about mechanisms regulating the intestinal fructose transporter GLUT5 in vivo. In neonatal rats, GLUT5 can be induced only by luminal fructose and only after 14 days of age, unless the gut is primed with dexamethasone prior to fructose perfusion. To elucidate the mechanisms underlying dexamethasone modulation of GLUT5 development, we first identified the receptor mediating its effects then determined whether those effects were genomic. The glucocorticoid receptor (GR) antagonist RU486 dose‐dependently prevented the dexamethasone‐mediated effects on body weight, intestinal arginase2 (a known GR‐regulated gene) and GLUT5. In contrast, an antagonist of the mineralocorticoid receptor as well as agonists of progesterone (PR) and pregnane‐X (PXR) receptors did not block the effects of dexamethasone. These receptor antagonists and agonists had no effect on the intestinal glucose transporter SGLT1. Translocation of the GR into the enterocyte nucleus occurred only in dexamethasone‐injected pups perfused with fructose, was accompanied by marked increases in brush border GLUT5 abundance, and was blocked by RU486. A priming duration of ∼24 h is optimal for induction but actinomycin D injection before dexamethasone priming prevented dexamethasone from allowing luminal fructose to induce GLUT5. Actinomycin D had no effect on dexamethasone‐independent fructose‐induced increases in glucose‐6‐phosphatase mRNA abundance, suggesting that it did not prevent fructose‐induction of GLUT5, but instead prevented dexamethasone‐induced synthesis of an intermediate required by fructose for GLUT5 regulation. In suckling rats < 14 days old, developmental regulation of transporters may involve cross‐talk between hormonal signals modulating intestinal maturation and nutrient signals regulating specific transporters.

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

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.—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, 4046‐4058 (2015). www.fasebj.org

Fructose-induced increases in expression of intestinal fructolytic and gluconeogenic genes are regulated by GLUT5 and KHK

Marked increases in fructose consumption have been tightly linked to metabolic diseases. One-third of ingested fructose is metabolized in the small intestine, but the underlying mechanisms regulating expression of fructose-metabolizing enzymes are not known. We used genetic mouse models to test the hypothesis that fructose absorption via glucose transporter protein, member 5 (GLUT5), metabolism via ketohexokinase (KHK), as well as GLUT5 trafficking to the apical membrane via the Ras-related protein in brain 11a (Rab11a)-dependent endosomes are required for the regulation of intestinal fructolytic and gluconeogenic enzymes. Fructose feeding increased the intestinal mRNA and protein expression of these enzymes in the small intestine of adult wild-type (WT) mice compared with those gavage fed with lysine or glucose. Fructose did not increase expression of these enzymes in the GLUT5 knockout (KO) mice. Blocking intracellular fructose metabolism by KHK ablation also prevented fructose-induced upregulation. Glycolytic hexokinase I expression was similar between WT and GLUT5- or KHK-KO mice and did not vary with feeding solution. Gavage feeding with the fructose-specific metabolite glyceraldehyde did not increase enzyme expression, suggesting that signaling occurs before the hydrolysis of fructose to three-carbon compounds. Impeding GLUT5 trafficking to the apical membrane using intestinal epithelial cell-specific Rab11a-KO mice impaired fructose-induced upregulation. KHK expression was uniformly distributed along the villus but was localized mainly in the basal region of the cytosol of enterocytes. The feedforward upregulation of fructolytic and gluconeogenic enzymes specifically requires GLUT5 and KHK and may proactively enhance the intestines ability to process anticipated increases in dietary fructose concentrations.