The effect of oxidative stress upon intestinal sugar transport: an in vitro study using human intestinal epithelial (Caco-2) cells

The pathogenesis of various gastrointestinal diseases, including gastrointestinal cancers and inflammatory bowel disease, is associated with increased oxidative stress levels. We aimed to investigate the effect of oxidative stress induced by tert-butylhydroperoxide (TBH) on the uptake of 3H-deoxy-D-glucose (3H-DG) and 14C-fructose by the human intestinal Caco-2 cell line. TBH (500 µM; 24 h) increased lipid peroxi- dation (TBARS) levels and was not cytotoxic. TBH (500 µM; 24 h) increased uptake of both low (SGLT1- mediated) and high concentrations (SGLT1- and GLUT2-mediated) of 3H-DG, but did not affect absorp- tion of 14C-fructose (GLUT2- and GLUT5-mediated). The polyphenol chrysin abolished the increase in TBARS levels and the increase in uptake of both low and high concentrations of 3H-DG induced by TBH. On the other hand, TBH blocked the inhibitory effect of chrysin on 14C-fructose uptake. 3H-DG uptake, but not 14C-fructose uptake, was sensitive to sweet taste receptor (STRs) inhibition (with lactisole). The inhibitory effect of lactisole in relation to uptake of 3H-DG (10 nM) (SGLT1-mediated), but not in relation to uptake of 3H-DG (50 mM) (SGLT1- and GLUT2-mediated), was abolished in the presence of TBH. So, these results show that the stimulatory effect of STRs on SGLT1-mediated transport is dependent on oxi- dative stress levels. In conclusion, this work shows that uptake of both 3H-DG and 14C-fructose is sensitive to oxidative stress levels. Moreover, it suggests that the three distinct transporters involved in the intestinal absorption of glucose and fructose (SGLT1, GLUT2 and GLUT5) have different sensitivities to oxidative stress levels, SGLT1 being the most sensitive and GLUT5 the least.

1.Introduction
Homeostatic control of the intestinal oxidative environment, i.e., a balance between reactive oxygen species (ROS) pro- duction and antioxidant defense systems1 is crucial to the function of this organ in nutrient digestion, absorption and immune response.2,3 Dysfunction in intestinal tissue caused by high oxidative stress levels is known to include loss of energy metabolism, gene mutations, impaired cellular trans- port mechanisms, and altered cell cycle and cell signaling.4,5 In line with this, various gastrointestinal pathological con- ditions, including gastrointestinal malignancies, gastroduode- nal ulcers, and inflammatory bowel disease (IBD) are associ- ated with increased oxidative stress levels.6The intestinal tract is exposed to high levels of reactive oxygen species (ROS). Besides ROS generated as by-products of normal cellular metabolic activities (namely mitochondrialrespiratory chain and the activities of enzymes such as NADPH oxidase, xanthine oxidase, cyclooxygenase, and NO synthase), multiple exogenous triggers induce oxidative stress at the intestinal levels. These include ultraviolet (UV) radiation, alcohol consumption, cigarette smoking, air pollutants, non- steroidal anti-inflammatory drugs and many other exogenous agents (e.g. Fe2+).2,5–10 Infections, ischemia-reperfusion (I/R) injury, and various inflammatory processes (including those induced by some food constituents and pathogens) also result in elevated levels of ROS in the intestine. Moreover, an inter- action between the intestinal epithelia and some groups of enteric commensal bacteria induce ROS generation.2 Finally, ingested nutrients such as proteins, sugars, fats, micronutri- ents, and food preservatives can generate ROS in the intes- tine.6 In relation to sugars, previous studies have shown that high-sugar diets and a fructose-rich diet cause increased oxi- dative stress at the intestinal level.

Of note, the oxidative effects of high-fructose feeding induced dysbiosis and corre- late with important changes in the gut microbial composition.16,17The main function of the small intestine is the absorption of nutrients. At the apical membrane of small intestinal epi- thelial cells, glucose is absorbed via sodium-dependentglucose co-transporter (SGLT1) and facilitative glucose trans- porter 2 (GLUT2 – a low-affinity glucose transporter).18–20 In relation to fructose, its transport across the apical cell mem- brane involves GLUT5 (facilitative glucose transporter 5 – a transporter specific for fructose) and GLUT2.18,21 After being taken up by intestinal epithelial cells, fructose and glucose are transported across the basolateral membrane by GLUT2 into the portal blood.21,22 The oxidative status across the gut lumen can influence nutrient intestinal epithelial uptake.5 However, few works have addressed the effect of oxidative stress on glucose cellular uptake,23 and nothing is known concerning the consequences of increased oxidative stress levels for the intestinal absorption of glucose and fructose.So, in the present study, we decided to investigate the effect of increased oxidative stress levels on fructose and glucose intestinal absorption and on the effect of two known modu- lators of sugar intestinal absorption, namely the sweet taste receptors (STRs) and the dietary polyphenol chrysin. Oxidative stress was induced with tert-butylhydroperoxide (TBH), a useful model compound to study mechanisms of oxidative stress injury. Proposed mechanisms of TBH-induced toxicity include alteration in intracellular calcium homeostasis follow- ing glutathione and protein thiol depletion, production of DNA strand breaks, lipid peroxidation and production of tert- butoxy radicals.

Although it has long been suggested that the gastrointesti- nal tract can sense the chemical composition of foods, only recently the nature of intestinal nutrient-sensing molecules and underlying mechanisms have been elucidated. STRs belong to the T1R family, included in the class C group of G-protein coupled receptors. In humans, the STR is a heterodi- mer comprised of T1R2 and T1R3 monomers.27 STRs can be activated by a wide range of chemically different compounds, including sugars (glucose, fructose, sucrose, maltose), artifi- cial sweeteners (e.g., saccharin, aspartame, cyclamate), sweet amino acids (D-tryptophan, D-phenylalanine, D-serine) and sweet proteins (monellin, brazzein, thaumatin).28,29 Previous works suggest that glucose sensing via STRs in the gastrointes- tinal tract regulates SGLT1- and GLUT2-mediated glucose absorption.30 However, nothing is known concerning the influ- ence of an oxidative stress setting on the activity of STRs. Moreover, the correlation between STRs and fructose intestinal absorption is unclear and needs more studies.31In relation to polyphenols, these are phytochemicals found abundantly in the human diet, namely in fruits, vegetables, tea, coffee, chocolates, legumes and cereals. These compounds have potential health benefits, which includes protection against oxidative stress, diabetes, cardiovascular disease, neu- rodegenerative disease, and aging.32–34 Several distinct poly- phenols are known to inhibit glucose intestinal absorp- tion,22,34 which could contribute to their beneficial effect on glucose homeostasis and, consequently, on obesity/type 2 dia- betes. Recently, a work made by our group showed that some polyphenols (chrysin, quercetin and apigenin) were able to inhibit fructose uptake by Caco-2 cells, by acting on the fruc- tose transporters GLUT2 and GLUT5.

2.Methods and materials
The Caco-2 cell line was obtained from Deutsche Sammlung von Mikroorganismen and Zellkulturen (Braunschweig, Germany) and was used between passage number 40–68. The cells were maintained in a humidified atmosphere of 5% CO2– 95% air and were grown in Minimum Essential Medium (Sigma, St Louis, MO) containing 5.55 mM glucose and sup-plemented with 15% fetal bovine serum, 25 mM HEPES, 100 units per ml penicillin, 100 μg ml−1 streptomycin, and 0.25 μg ml−1 amphotericin B (all from Sigma). Culture medium was changed every 3–4 days and the culture was split every 12 days.For sub-culturing, the cells were removed enzymatically (0.25% trypsin–EDTA, 5 min, 37 °C), split 1 : 3, and subcultured in plastic culture dishes (21 cm2; ∅ 60 mm; Corning Costar,Corning, NY).For uptake studies, Caco-2 cells were seeded on 24-well plastic cell culture clusters (2 cm2; ∅ 16 mm; Corning Costar), and the experiments were performed 10–14 days after the initialseeding (100% confluence). The culture medium was made free of fetal bovine serum for 24 h before the experiments. The uptake experiments were performed with Caco-2 cells incu- bated in glucose-free Krebs buffer (containing, in mM: 125 NaCl, 4.8 KCl, 1.2 MgSO4, 1.2 CaCl2, 25 NaHCO3, 1.6 KH2PO4,0.4 K2HPO4, and 20 HEPES ( pH 7.4)). Initially, the culture medium was aspirated and the cells were washed with 0.3 ml buffer at 37 °C. Then, the cell monolayers were preincubated for 20 min in 0.3 ml buffer at 37 °C. Uptake was initiated by the addition of 0.3 ml medium at 37 °C containing 14C-fruc- tose (100 nM, 10 µM or 50 mM) or 3H-DG (10 nM, 10 µM or 50 mM). Incubation was stopped after 6 min by removing the incubation medium, placing the cells on ice, and rinsing the cells with 0.5 ml ice-cold buffer.

The cells were then solubil- ized with 0.3 ml 0.1% (v/v) Triton X-100 (in 5 mM Tris HCl, pH 7.4) and placed at 37 °C overnight. Radioactivity in the cells was measured by liquid scintillation counting.The short-term effect of compounds on 14C-fructose and 3H-DG uptake was tested by preincubating (20 min) and incu- bating (6 min) cells with 14C-fructose or 3H-DG in the presence of the compounds to be tested (or the respective solvent).The long-term effect of compounds on 14C-fructose and 3H-DG uptake was tested by cultivating 10–13-day-old cell cul- tures in culture medium without fetal bovine serum in the presence of the compounds to be tested (or the respective solvent). The transport experiments were performed after 24 h of treatment. The transport experiments were identical to the experiments described above, and compounds (or solvent) were also present during the preincubation (20 min) and incu- bation (6 min) periods.In order to induce oxidative stress, Caco-2 cells were treated with TBH (500 μM, dissolved in decane), for 24 h, in the serum-free culture medium. Control cells were exposed to anidentical concentration of decane (1% (v/v)) in the serum-free culture medium.Effect of TBH on cell viability. At the end of the 24 h treatment period with TBH (or the respective solvent), cell via- bility was assessed by quantification of extracellular LDH activity. Cellular leakage of LDH into the extracellular culture medium was determined spectrophotometrically by measuring the decrease in absorbance of NADH during the reduction of pyruvate to lactate, as described.25 The LDH activity is expressed as the percentage of extracellular activity in relation to total cellular LDH activity.

To determine total cellular LDH activity, control cells were exposed to 0.1% (v/v) Triton X-100 for 30 min at 37 °C.Effect of TBH on culture growth – sulphorhodamine B (SRB) assay. Caco-2 cells were treated with TBH (or the respective solvent) during 24 h. Then, the cells were incubated with 50% trichloroacetic acid for 1 h at 4 °C. Each well was then washed with tap water to remove trichloroacetic acid and air-dried for 30 min 0.4% SRB dissolved in 1% acetic acid was added and maintained for 15 min. Afterwards, cell cultures were washed with 1% trichloroacetic acid to remove residual dye and again air-dried. Stained cells were then solubilized 10 mM Tris·NaOH solution ( pH 10.5) and the absorbance of each well was determined at 540 nm.32,33Effect of TBH on lipid peroxidation (malondialde- hyde) levels. The formation of thiobarbituric acid-reactive sub- stances (TBARS assay) is used as a lipid peroxidation (oxidative stress) biomarker. At the end of the 24h-treatment period of Caco-2 cells with TBH (or the respective solvent), the reaction was started by addition of 50% acetic acid to each sample, followed by a centrifugation for 2 min at 6000 rpm. Then, 1% 2-thiobarbituric acid was added to the supernatant and the reaction was carried out in a boiling water bath for 40 min. A pink-colored complex was quantified spectrophotometrically at 535 nm.31,32The protein content of cell monolayers was determined as described by Bradford, using human serum albumin as standard.35Arithmetic means are given with SEM, and geometric means are given with 95% confidence limits.

The value of n indicates the number of replicates of at least 2 different experiments. Statistical significance of the difference between two groups was evaluated by Student’s t-test; statistical analysis of the difference between various groups was evaluated by the ana- lysis of variance (ANOVA) test, followed by the Bonferroni test. Differences were considered to be significant when P < 0.05.All chemicals were obtained from standard commercial suppli- ers and were of analytical-grade quality. 14C-D-Fructose (fructose,D-[14C(U)]; (14C-FRU); specific activity 250–360 mCi mmol−1);[1,2-3H(N)]-deoxy-D-glucose; (3H-DG); specific activity 60 mCi mmol−1 (American Radiolabeled Chemicals, St Louis, MO, USA), antibiotic/antimycotic solution (100 U ml−1 penicillin; 100 mg ml−1streptomycin and 0.25 mg ml−1 amphotericin B), MEM medium, HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfo-nic acid), NADH (reduced nicotinamide adenine dinucleotide), chrysin, D-glucose, D-fructose, trypsin–EDTA (ethylenediamine tetraacetic acid) solution, tert-butylhydroperoxide (TBH), decane, sulforhodamine B (SRB), 2-thiobarbituric acid, trichloroacetic acid sodium salt, sodium pyruvate, malondialdehyde (Sigma, St Louis, MO, USA); DMSO (dimethylsulphoxide), Triton X-100 (Merck, Darmstadt, Germany); fetal bovine serum (Invitrogen Corporation, Carlsbad, CA, USA); lactisole (2-(4-methoxy- phenoxy)-propanoic acid, monosodium salt) (Cayman, MI, USA). The drugs to be tested were dissolved in water, decane or DMSO, the final concentration of these solvents being 1% in the buffer or 0.1% in the culture medium for short- and long- term treatments, respectively. Controls for these drugs were run in the presence of the solvent. None of these solvents sig- nificantly affected any of the measured parameters (14C-fruc- tose and 3H-DG uptake, cell viability, proliferation and lipidperoxidation; data not shown).

3.Results
In a first series of experiments, we investigated the effect of a 24 h-exposure to TBH (100–3000 µM) on Caco-2 cell viability (assessed with the LDH assay; Fig. 1A) and culture growth (assessed with the sulforhodamine B assay; Fig. 1B).Fig. 1A shows that TBH up to 500 μM did not induce a cyto-toxic effect upon this cell line. However, when cells were exposed to higher concentrations of TBH (1000 and 3000 μM), a very marked and concentration-dependent cytotoxic effect wasfound. In agreement with these findings, exposure of Caco-2 cells to TBH up to 500 μM did not cause any significant effect on culture growth, but the exposure of the cells to higher con- centrations of TBH (1000 and 3000 μM) caused a strong inhibi- tory effect on culture growth (Fig. 1B). To confirm that TBHinduced oxidative stress in Caco-2 cells, we measured the gene- ration of lipid peroxidation products. Fig. 1C shows that 500 μM TBH (24 h) caused a significant increase in TBARS levels inCaco-2 cells. From these results, a non-cytotoxic and non- growth inhibitory concentration of TBH able to induce oxidative stress (500 μM) was chosen for the remainder experiments.The effect of oxidative stress induced by TBH (500 µM; 24 h)on 3H-DG and 14C-fructose uptake by Caco-2 cells was next investigated. As shown in Fig. 2, TBH caused a marked increase in 3H-DG (10 nM and 10 µM) uptake. In contrast, TBH had no significant effect upon 14C-fructose uptake (Fig. 3). These results suggest that an increased oxidative stress environment increases glucose uptake by intestinal cells, but has no effect on fructose uptake.3.2.2.Influence of chrysin on the effect of TBH. In these experiments, we tested if the effect of TBH on sugar uptake is influenced by chrysin. This dietary polyphenol (100 μM; 24 h)showed a ±15% inhibitory effect on the uptake of 10 nM3H-DG, but uptake of a higher concentration of glucose (10 µM) was not affected (Fig. 2). When combined with TBH, chrysin was able to abolish the stimulatory effect of TBH on the uptake of both concentrations of 3H-DG (10 nM and 10 µM) (Fig. 2). So, this dietary polyphenol appears to be capable of inhibiting the increase in 3H-DG uptake induced by TBH. Interestingly enough, when Caco-2 cells were exposed tochrysin (100 μM), the TBARS levels were reduced when com-pared to control, even when TBH was also present (Fig. 1C).

So, the abolition of the stimulatory effect of TBH on 3H-DG uptake by chrysin may be mediated by a reduction in oxidative stress levels.In relation to 14C-fructose (100 nM and 10 µM) uptake, chrysin was found to reduce it by ±25%. However, when testedin combination with TBH 500 μM, the inhibitory effect of chrysin disappeared (Fig. 3). These results suggest thatincreased oxidative stress levels blocked the effect of chrysin on the 14C-fructose uptake. Altogether, these results suggest that oxidative stress levels influence both glucose and fructose uptake by Caco-2 cells, as an increase (TBH-mediated) and a decrease (chrysin-mediated) in oxidative stress levels have opposite effects on the cellular uptake of both sugars.Effect of lactisole on 3H-DG and 14C-fructose uptake. To investigate the influence of STRs in 3H-DG and 14C-fructose uptake by Caco-2 cells, we tested both the short- (20 min) and long-term (24 h) effect of several concentrations of the STRs inhibitor lactisole (0.1, 0.25, 0.5 mM) on the uptake of distinctconcentrations of 3H-DG (10 nM, 10 μM and 50 mM) and 14C-fructose (100 nM, 10 µM and 50 mM).Fig. 4A and C demonstrate that, in the short-term, lactisole did not show any effect on 3H-DG uptake by Caco-2 cells. In contrast, a longer (24 h) exposure to this STRs inhibitor (0.5 mM) was able to significantly reduce low (10 nM) and high (50 mM) 3H-DG uptake (Fig. 4B and E).

So, these results suggest that glucose uptake by Caco-2 cells is regulated by STRs.As to 14C-fructose, apart from a stimulatory effect found after a short-term exposure to lactisole on the uptake of 14C- fructose 10 µM, this drug was not able to affect uptake of thissugar (Fig. 5). For this reason, the influence of lactisole on the effect of TBH was investigated in relation to 3H-DG uptake only (see below).In agreement with the results pre- sented above (Fig. 3), TBH was able to significantly enhance uptake of 10 µM 3H-DG (Fig. 6A). However, when a higher concentration of 3H-DG was used, TBH showed no effect (Fig. 6B).Influence of lactisole on the effect of TBH. In this final series of experiments, we investigated if the effect of TBH on 3H-DG by Caco-2 cells is influenced by the presence of lacti- sole (100 μM). In agreement with previous findings (Fig. 4), along-term exposure of the cells to lactisole (100 μM) reduced3H-DG uptake by 20% (Fig. 6). We verified that the inhibitory effect of lactisole in relation to uptake of 3H-DG (10 nM) was abolished in the presence of increased oxidative stress levels (Fig. 6A). In contrast, the inhibitory effect of this compound in relation to uptake of a higher concentration of 3H-DG (50 mM) was not modified in the presence of TBH (Fig. 6B). So, we con- clude that the influence of STRs in relation to uptake of a low concentration of glucose (but not in relation to a high concen- tration of glucose) is dependent on oxidative stress levels.

4.Discussion
Several studies have demonstrated that ROS can interfere with the activity of membrane transporters.36–38 However, nothing is known in relation to the effect of oxidative stress upon the intestinal absorption of sugars. So, the aim of this work was to investigate the effect of oxidative stress on 3H-DG and 14C-fruc- tose uptake by Caco-2 cells. The human intestinal Caco-2 cell line, originally obtained from a human colon adenocarcinoma, has been extensively used as a model of the intestinal barrier.39Oxidative stress was generated with TBH, a useful model compound to study mechanisms of oxidative stress injury.40–43 A previous study from our group demonstrated a cytotoxic and antiproliferative effect of TBH in Caco-2 cells, associated with increased levels of oxidative stress biomarkers.25,26In the first series of experiments, we confirmed TBH as an oxidative stress-inducing compound in our cell culture model, as exposure of these cells to TBH (500 µM) caused a significant increase in MDA levels. This enhance in lipid peroxidation is considered a hallmark of oxidative stress and has been associ- ated with TBH-induced toxicity.44Contrary to what was observed after exposure of the cells to TBH 1000 and 3000 μM, exposure of Caco-2 cells to TBH 500 μM did not appear to cause significant cytotoxicity. Basedon these results, we concluded that Caco-2 cells exposed to 500 μM TBH for 24 h constitute a good cellular model to inves- tigate the effects of oxidative stress upon intestinal uptake ofsugars, and this concentration of TBH was used in subsequent experiments.

We verified, in the presence of TBH, a marked increase (30–50%) in the uptake of the lowest concentrations of 3H-DG(10 nM and 10 μM) (mainly SGLT1-mediated). On the otherhand, absorption of a higher concentration of 3H-DG (50 mM) (GLUT-2 mediated) and of 14C-fructose (GLUT2- and GLUT5- mediated) was not affected by exposure to TBH. Taken together, these effects support the hypothesis that oxidative stress is affecting predominantly SGLT1, with no effect on GLUT2 and GLUT5. In agreement with this hypothesis, a con- dition associated with an increased oxidative environment (type 2 diabetes) induced an increase in the expression levels of SGLT1.45 Of note, increased oxidative stress has been pre- viously described to have a stimulatory effect not only on SGLT1, but also on SGLT2 45,46 and in glucose membrane transporters of the GLUT family, including GLUT2.47,48 However, oxidative stress or oxidative stress-associated con-ditions have been also found to have a negative impact in GLUT transporters, including GLUT2 expression49,50 or to have no impact at all.24,51 Of note, in some of these studies, the direct effect of oxidative stress was not investigated; rather, the consequences of conditions known to be associated with increased oxidative stress levels were evaluated; moreover, the functional activity of these transporters was not always evalu- ated. In this context, the present study gives a more direct information on the relationship between oxidative stress and membrane transport activity.Dietary polyphenols are emerging as prophylactic and therapeutic agents against metabolic disorders involving free radicals in their pathogenesis, especially at the GT level. These compounds aid to protect the intestine against damage by ROS present in foods or generated within the GT.52Interestingly, in this study, chrysin (500 μM) was able to reducesignificantly TBARS levels, when compared with control, even when this polyphenol was combined with TBH. So, chrysin proved to be very effective as an antioxidative agent.In the present work, chrysin was found to inhibit uptake of 3H-DG (10 nM) and 14C-fructose (100 nM and 10 µM).

So, chrysin might be effective in inhibiting the intestinal absorp- tion of these two sugars. To our knowledge, this is the first description of an inhibitory effect of chrysin upon 3H-DG intestinal absorption, although the inhibitory effect of chrysin towards 14C-fructose uptake confirms a previous work by our group.32 In that work, chrysin was found to decrease expression of both GLUT2 and GLUT5, leading us to conclude, from its inhibitory effect upon GLUT2, that chrysin would also be able to reduce glucose uptake by these cells. This assump- tion was confirmed in the present work. However, it must be stressed out that the effect of chrysin upon SGLT1 activity or expression is not known, and it is possible that this polyphe- nol is also acting on this glucose transporter. Because the inhibitory effect of chrysin on 3H-DG uptake was observed only with the lowest concentration of 3H-DG tested, where the con- tribution of SGLT1 for glucose uptake is expected to be greater (due to its higher affinity for this sugar than GLUT2),53 we do not only suggest that chrysin inhibits SGLT1, but also that it has a more marked inhibitory effect upon SGLT1 than upon GLUT2.Analysis of the effect of chrysin conjugated with TBH on 3H-DG and 14C-fructose uptake led us to conclude that oxi- dative stress levels influence both glucose and fructose uptake by Caco-2 cells, as an increase (TBH-mediated) and a decrease (chrysin-mediated) in oxidative stress levels have opposite effects on the cellular uptake of both sugars.Almost nothing is known concerning the effect of oxidative stress on glucose or fructose intestinal uptake. Based on the effect of TBH and chrysin on TBARS levels, our results are compatible with the fact that lipid peroxidation induced by TBH induces an increase in glucose uptake, and that chrysin, by reducing lipid peroxidation levels, counteracts this stimu- latory effect of TBH.

This hypothesis is also supported by a pre- vious work using cultured cardiomyocytes, where it was con- cluded that lipid peroxidation evokes an adaptive responseresulting in an increased glucose uptake, presumably to restore cellular energy, which was also inhibited by an anti- oxidant (vitamin E).48 However, based on the opposite effect of TBH and chrysin on SGLT1-mediated glucose transport, it is possible that the influence of oxidative stress levels on SGLT1- mediated transport will also contribute to the effect of TBH on the 3H-DG uptake. There is a huge gap of works addressing oxidative stress and sugars transport into the intestine and much more studies are required.Another point addressed in this study was to investigate the influence of STRs on the intestinal absorption of sugars, and the putative interference of increased oxidative stress levels with this effect. For this, we first tested the effect of lactisole (a STRs inhibitor) on the uptake of different concentrations of 3H-DG and 14C-fructose. A recent study showed that T1R2 and T1R3 proteins are present in Caco-2 cell membranes.54Although short-term exposure to lactisole had no effect on 3H-DG uptake, long-term (24 h) exposure to lactisole (0.5 mM) caused a significant reduction on the uptake of both a low (10 nM) and a high (50 mM) concentration of 3H-DG. These find- ings indicate that glucose uptake is upregulated by STRs in Caco-2 cell. This observation is corroborated by prior works showing that activation of STRs on the apical membrane of enterocytes enhance small intestinal glucose transport capacity, mediated by a rapid and reversible insertion of GLUT2 into the apical membrane, and also by an increase in SGLT1 expression and activity.21,54 Additionally, the obser- vation that upregulation of SGLT1 and GLUT2 is absent in SRTs knockout rodents indicates that a strong correlation exists between STRs activation and modulation of glucose transporter expression and activity.20,55On the other hand, lactisole exposure did not show a con- sistent effect on 14C-fructose uptake.

Indeed, apart from a short-term stimulatory effect on uptake of 14C-fructose(10 μM), long-term exposure to lactisole had no effect at all onthe 14C-fructose uptake. Although it is known that fructose is able to activate STRs,54 there is not clear information about the regulation of fructose uptake by STRs. A recent research suggests that STRs play a role in modulating glucose, but not fructose, uptake, because when glucose/fructose were adminis- tered together, there was an effect of lactisole on glucose, but not fructose, transport, possibly mediated by T1R3.54 However, lactisole showed an effect contrary to the expected, i.e., it increased glucose uptake, and showed per se no effect on glucose and fructose uptake ( probably because a short-term exposure (3 h) was chosen).54Because lactisole had a more consistent effect in relation to3H-DG uptake, in the last part of this work, we investigated whether the effect of lactisole (500 μM) on 3H-DG (10 μM and 50 mM) uptake is influenced by TBH (500 μM). A long-term exposure to lactisole markedly reduced uptake of a low (mainlySGLT1-mediated) and high (mainly GLUT2-mediated) 3H-DG uptake. This is in agreement with the stimulatory effect pre- viously described for STRs on SGLT1 and GLUT2 (see above).Interestingly enough, the inhibitory effect of lactisole on the uptake of low 3H-DG (10 μM) was abolished by TBH, but TBH did not change the inhibitory effect of lactisole on the uptakeof high concentration of 3H-DG (50 mM). This is consistent with our previous conclusion that TBH inhibits SGLT1 (low affinity), but not GLUT2 (low affinity)-mediated 3H-DG uptake.

5.Conclusion
This work shows that uptake of both 3H-DG and 14C-fructose are sensitive to oxidative stress levels. However, it suggests that the three distinct transporters involved in the intestinal absorp- tion of glucose and fructose (SGLT1, GLUT2 and GLUT5) have different sensitivities to oxidative stress levels, SGLT1 being the most sensitive and GLUT5 the least. Our results also show that the stimulatory effect of STRs on SGLT1-mediated transport is dependent on oxidative stress levels (Table 1). So, more research is needed in order to Mizagliflozin determine the precise role of oxi- dative stress upon sugar intestinal absorption.