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Elevated reactive oxygen species (ROS) in type 2 diabetes cause cellular damage in many organs. Recently, the new class of glucose-lowering agents, SGLT-2 inhibitors, have been shown to reduce the risk of developing diabetic complications; however, the mechanisms of such beneficial effect are largely unknown. Here we aimed to investigate the effects of dapagliflozin on cell proliferation and cell death under oxidative stress conditions and explore its underlying mechanisms. Human proximal tubular cells (HK-2) were used. Cell growth and death were monitored by cell counting, water-soluble tetrazolium-1 (WST-1) and lactate dehydrogenase (LDH) assays, and flow cytometry. The cytosolic and mitochondrial (ROS) production was measured using fluorescent probes (H2DCFDA and MitoSOX) under normal and oxidative stress conditions mimicked by addition of H2O2. Intracellular Ca2+ dynamics was monitored by FlexStation 3 using cell-permeable Ca2+ dye Fura-PE3/AM. Dapagliflozin (0.1-10 μM) had no effect on HK-2 cell proliferation under normal conditions, but an inhibitory effect was seen at an extreme high concentration (100 μM). However, dapagliflozin at 0.1 to 5 μM showed remarkable protective effects against H2O2-induced cell injury via increasing the viable cell number at phase G0/G1. The elevated cytosolic and mitochondrial ROS under oxidative stress was significantly decreased by dapagliflozin. Dapagliflozin increased the basal intracellular [Ca2+]i in proximal tubular cells, but did not affect calcium release from endoplasmic reticulum and store-operated Ca2+ entry. The H2O2-sensitive TRPM2 channel seemed to be involved in the Ca2+ dynamics regulated by dapagliflozin. However, dapagliflozin had no direct effects on ORAI1, ORAI3, TRPC4 and TRPC5 channels. Our results suggest that dapagliflozin shows anti-oxidative properties by reducing cytosolic and mitochondrial ROS production and altering Ca2+ dynamics, and thus exerts its protective effects against cell damage under oxidative stress environment., Competing Interests: No authors have competing interests.

LEFTY2 (endometrial bleeding associated factor; EBAF or LEFTYA), a cytokine released shortly before menstrual bleeding, is a negative regulator of cell proliferation and tumour growth. LEFTY2 down-regulates Na+/H+ exchanger activity with subsequent inhibition of glycolytic flux and lactate production in endometrial cancer cells. Glucose can be utilized not only for glycolysis but also for glycogen formation. Both glycolysis and glycogen formation require cellular glucose uptake which could be accomplished by the Na+ coupled glucose transporter-1 (SGLT1; SLC5A1). The present study therefore explored whether LEFTY2 modifies endometrial SGLT1 expression and activity as well as glycogen formation. Ishikawa and HEC1a cells were exposed to LEFTY2, SGLT1 and glycogen synthase (GYS1) transcript levels determined by qRT-PCR. SGLT1, GYS1 and phospho-GYS1 protein abundance was quantified by western blotting, cellular glucose uptake from 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) uptake, and cellular glycogen content utilizing an enzymatic assay and subsequent colorimetry. As a result, a 48-hour treatment with LEFTY2 significantly increased SGLT1 and GYS1 transcript levels as well as SGLT1 and GYS1 protein abundance in both Ishikawa and HEC1a cells. 2-NBDG uptake and cellular glycogen content were upregulated significantly in Ishikawa (type 1) but not in type 2 endometrial HEC1a cells, although there was a tendency of increased 2-NBDG uptake. Further, none of the effects were seen in human benign endometrial cells (HESCs). Interestingly, in both Ishikawa and HEC1a cells, a co-treatment with TGF-β reduced SGLT1, GYS and phospho-GYS protein levels, and thus reduced glycogen levels and again HEC1a cells had no significant change. In conclusion, LEFTY2 up-regulates expression and activity of the Na+ coupled glucose transporter SGLT1 and glycogen synthase GYS1 in a cell line specific manner. We further show the treatment with LEFTY2 fosters cellular glucose uptake and glycogen formation and TGF-β can negate this effect in endometrial cancer cells., Competing Interests: The authors have declared that no competing interests exist.

Respiratory infection can be exacerbated by the high glucose concentration in the airway surface liquid (ASL). We investigated the effects of salbutamol and phlorizin on the pulmonary function, oxidative stress levels and SGLT1 activity in lung, pulmonary histopathological damages and survival rates of rats with sepsis. Sepsis was induced by cecal ligation and puncture surgery (CLP). Twenty-four hours after surgery, CLP rats were intranasally treated with saline, salbutamol or phlorizin. After 2 hours, animals were anesthetized and sacrificed. Sepsis promoted atelectasis and bronchial inflammation, and led to increased expression of SGLT1 on cytoplasm of pneumocytes. Salbutamol treatment reduced bronchial inflammation and promoted hyperinsuflation in CLP rats. The interferon-ɤ and Interleucin-1β concentrations in bronchoalveolar lavage (BAL) were closely related to the bronchial inflammation regulation. Salbutamol stimulated SGLT1 in plasma membrane; whereas, phlorizin promoted the increase of SGLT1 in cytoplasm. Phlorizin reduced catalase activity and induced a significant decrease in the survival rate of CLP rats. Taken together, sepsis promoted atelectasis and lung inflammation, which can be associated with SGLT1 inhibition. The loss of function of SGLT1 by phlorizin are related to the augmented disease severity, increased atelectasis, bronchial inflammation and a significant reduction of survival rate of CLP rats. Alternatively salbutamol reduced BAL inflammatory cytokines, bronchial inflammation, atelectasis, and airway damage in sepsis. These data suggest that this selective β2-adrenergic agonist may protect lung of septic acute effects., Competing Interests: The authors have declared that no competing interests exist.

Flavanoids and related polyphenols, among them hesperitin, have been shown to modulate cellular glucose transport by targeting SGLT-1 and GLUT-2 transport proteins. We aimed to investigate whether homoeriodictyol, which is structurally related to hesperitin, affects glucose uptake in differentiated Caco-2 cells as a model for the intestinal barrier. The results revealed that, in contrast to other polyphenols, the flavanon homoeriodictyol promotes glucose uptake by 29.0 ± 3.83% at a concentration of 100 μM. The glucose uptake stimulating effect was sensitive to phloridzin, but not to phloretin, indicating an involvement of the sodium-coupled glucose transporter SGLT-1, but not of sodium-independent glucose transporters (GLUT). In addition, in contrast to the increased extracellular serotonin levels by stimulation with 500 mM D-(+)-glucose, treatment with 100 μM homoeriodictyol decreased serotonin release by -48.8 ± 7.57% in Caco-2 cells via a phloridzin-sensitive signaling pathway. Extracellular serotonin levels were also reduced by -57.1 ± 5.43% after application of 0.01 μM homoeriodictyol to human neural SH-SY5Y cells. In conclusion, we demonstrate that homoeriodictyol affects both the glucose metabolism and the serotonin system in Caco-2 cells via a SGLT-1-meditated pathway. Furthermore, the results presented here support the usage of Caco-2 cells as a model for peripheral serotonin release. Further investigations may address the value of homoeriodictyol in the treatment of anorexia and malnutrition through the targeting of SGLT-1., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: The authors K. Geissler, J. Hans, J.P. Ley, and G.E. Krammer are employees at Symrise AG, Holzminden, Germany. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Transmissible gastroenteritis virus (TGEV) is a coronavirus that causes villus atrophy, followed by crypt hyperplasia, reduces the activities of intestinal digestive enzymes, and disrupts the absorption of intestinal nutrients. In vivo, TGEV primarily targets and infects intestinal epithelial cells, which play an important role in glucose absorption via the apical and basolateral transporters Na+-dependent glucose transporter 1 (SGLT1) and facilitative glucose transporter 2 (GLUT2), respectively. In this study, we therefore sought to evaluate the effects of TGEV infection on glucose uptake and SGLT1 and GLUT2 expression. Our data demonstrate that infection with TGEV resulted in increased glucose uptake and augmented expression of EGFR, SGLT1 and GLUT2. Moreover, inhibition studies showed that EGFR modulated glucose uptake in control and TGEV infected cells. Finally, high glucose absorption was subsequently found to promote TGEV replication., Competing Interests: The authors have declared that no competing interests exist.

Na-coupled cotransporters are proteins that use the trans-membrane electrochemical gradient of Na to activate the transport of a second solute. The sodium-glucose cotransporter 1 (SGLT1) constitutes a well-studied prototype of this transport mechanism but essential molecular characteristics, namely its quaternary structure and the exact arrangement of the C-terminal transmembrane segments, are still debated. After expression in Xenopus oocytes, human SGLT1 molecules (hSGLT1) were labelled on an externally accessible cysteine residue with a thiol-reactive fluorophore (tetramethylrhodamine-C5-maleimide, TMR). Addition of dipicrylamine (DPA, a negatively-charged amphiphatic fluorescence "quencher") to the fluorescently-labelled oocytes is used to quench the fluorescence originating from hSGLT1 in a voltage-dependent manner. Using this arrangement with a cysteine residue introduced at position 624 in the loop between transmembrane segments 12 and 13, the voltage-dependent fluorescence signal clearly indicated that this portion of the 12-13 loop is located on the external side of the membrane. As the 12-13 loop begins on the intracellular side of the membrane, this suggests that the 12-13 loop is re-entrant. Using fluorescence resonance energy transfer (FRET), we observed that different hSGLT1 molecules are within molecular distances from each other suggesting a multimeric complex arrangement. In agreement with this conclusion, a western blot analysis showed that hSGLT1 migrates as either a monomer or a dimer in reducing and non-reducing conditions, respectively. A systematic mutational study of endogenous cysteine residues in hSGLT1 showed that a disulfide bridge is formed between the C355 residues of two neighbouring hSGLT1 molecules. It is concluded that, 1) hSGLT1 is expressed as a disulfide bridged homodimer via C355 and that 2) a portion of the intracellular 12-13 loop is re-entrant and readily accessible from the extracellular milieu.

Objective: Sodium-glucose cotransporter 1 (SGLT1) is thought to be expressed in the heart as the dominant isoform of cardiac SGLT, although more information is required to delineate the subtypes of SGLTs in human hearts. Moreover, the functional role of SGLTs in the heart remains to be fully elucidated. We herein investigated whether SGLT1 is expressed in human hearts and whether SGLTs significantly contribute to cardiac energy metabolism during ischemia-reperfusion injury (IRI) via enhanced glucose utilization in mice., Methods and Results: We determined that SGLT1 was highly expressed in both human autopsied hearts and murine perfused hearts, as assessed by immunostaining and immunoblotting with membrane fractionation. To test the functional significance of the substantial expression of SGLTs in the heart, we studied the effects of a non-selective SGLT inhibitor, phlorizin, on the baseline cardiac function and its response to ischemia-reperfusion using the murine Langendorff model. Although phlorizin perfusion did not affect baseline cardiac function, its administration during IRI significantly impaired the recovery in left ventricular contractions and rate pressure product, associated with an increased infarct size, as demonstrated by triphenyltetrazolium chloride staining and creatine phosphokinase activity released into the perfusate. The onset of ischemic contracture, which indicates the initiation of ATP depletion in myocardium, was earlier with phlorizin. Consistent with this finding, there was a significant decrease in the tissue ATP content associated with reductions in glucose uptake, as well as lactate output (indicating glycolytic flux), during ischemia-reperfusion in the phlorizin-perfused hearts., Conclusions: Cardiac SGLTs, possibly SGLT1 in particular, appear to provide an important protective mechanism against IRI by replenishing ATP stores in ischemic cardiac tissues via enhancing availability of glucose. The present findings provide new insight into the significant role of SGLTs in optimizing cardiac energy metabolism, at least during the acute phase of IRI.

Oral glucose supply is important for neonatal calves to stabilize postnatal plasma glucose concentration. The objective of this study was to investigate ontogenic development of small intestinal growth, lactase activity, and glucose transporter in calves (n = 7 per group) that were born either preterm (PT; delivered by section 9 d before term) or at term (T; spontaneous vaginal delivery) or spontaneously born and fed colostrum for 4 days (TC). Tissue samples from duodenum and proximal, mid, and distal jejunum were taken to measure villus size and crypt depth, protein concentration of mucosa and brush border membrane vesicles (BBMV), total DNA and RNA concentration of mucosa, mRNA expression and activity of lactase, and mRNA expression of sodium-dependent glucose co-transporter-1 (SGLT1) and facilitative glucose transporter 2 (GLUT2) in mucosal tissue. Additionally, protein expression of SGLT1 in BBMV and GLUT2 in crude mucosal membranes and immunochemical localization of GLUT2 in the enterocytes were determined. Villus height in distal jejunum was lower in TC than in T. Crypt depth in all segments was largest and the villus height/crypt depth ratio in jejunum was smallest in TC calves. Concentration of RNA was highest in duodenal mucosa of TC calves, but neither lactase mRNA and activity nor SGLT1 and GLUT2 mRNA and protein expression differed among groups. Localization of GLUT2 in the apical membrane was greater, whereas in the basolateral membrane was lower in TC than in T and PT calves. Our study indicates maturation processes after birth for mucosal growth and trafficking of GLUT2 from the basolateral to the apical membrane. Minor differences of mucosal growth, lactase activity, and intestinal glucose transporters were seen between PT and T calves, pointing at the importance of postnatal maturation and feeding for mucosal growth and GLUT2 trafficking.

We hypothesize that glucosensor mechanisms other than that mediated by glucokinase (GK) operate in hypothalamus and hindbrain of the carnivorous fish species rainbow trout and stress affected them. Therefore, we evaluated in these areas changes in parameters which could be related to putative glucosensor mechanisms based on liver X receptor (LXR), mitochondrial activity, sweet taste receptor, and sodium/glucose co-transporter 1 (SGLT-1) 6 h after intraperitoneal injection of 5 mL x Kg(-1) of saline solution alone (normoglycaemic treatment) or containing insulin (hypoglycaemic treatment, 4 mg bovine insulin x Kg(-1) body mass), or D-glucose (hyperglycaemic treatment, 500 mg x Kg(-1) body mass). Half of tanks were kept at a 10 Kg fish mass x m(-3) and denoted as fish under normal stocking density (NSD) whereas the remaining tanks were kept at a stressful high stocking density (70 kg fish mass x m(-3)) denoted as HSD. The results obtained in non-stressed rainbow trout provide evidence, for the first time in fish, that manipulation of glucose levels induce changes in parameters which could be related to putative glucosensor systems based on LXR, mitochondrial activity and sweet taste receptor in hypothalamus, and a system based on SGLT-1 in hindbrain. Stress altered the response of parameters related to these systems to changes in glycaemia.

Intestinal glucose absorption is mediated by SGLT1 whereas GLUT2 is considered to provide basolateral exit. Recently, it was proposed that GLUT2 can be recruited into the apical membrane after a high luminal glucose bolus allowing bulk absorption of glucose by facilitated diffusion. Moreover, SGLT1 and GLUT2 are suggested to play an important role in intestinal glucose sensing and incretin secretion. In mice that lack either SGLT1 or GLUT2 we re-assessed the role of these transporters in intestinal glucose uptake after radiotracer glucose gavage and performed Western blot analysis for transporter abundance in apical membrane fractions in a comparative approach. Moreover, we examined the contribution of these transporters to glucose-induced changes in plasma GIP, GLP-1 and insulin levels. In mice lacking SGLT1, tissue retention of tracer glucose was drastically reduced throughout the entire small intestine whereas GLUT2-deficient animals exhibited higher tracer contents in tissue samples than wild type animals. Deletion of SGLT1 resulted also in reduced blood glucose elevations and abolished GIP and GLP-1 secretion in response to glucose. In mice lacking GLUT2, glucose-induced insulin but not incretin secretion was impaired. Western blot analysis revealed unchanged protein levels of SGLT1 after glucose gavage. GLUT2 detected in apical membrane fractions mainly resulted from contamination with basolateral membranes but did not change in density after glucose administration. SGLT1 is unequivocally the prime intestinal glucose transporter even at high luminal glucose concentrations. Moreover, SGLT1 mediates glucose-induced incretin secretion. Our studies do not provide evidence for GLUT2 playing any role in either apical glucose influx or incretin secretion.

Polyphenols contained within plant tissues are consumed in significant amounts in the human diet and are known to influence a number of biological processes. This study investigated the effects of an anthocyanin-rich berry-extract on glucose uptake by human intestinal Caco-2 cells. Acute exposure (15 min) to berry extract (0.125%, w/v) significantly decreased both sodium-dependent (Total uptake) and sodium-independent (facilitated uptake) ³H-D-glucose uptake. In longer-term studies, SGLT1 mRNA and GLUT2 mRNA expression were reduced significantly. Polyphenols are known to interact directly with glucose transporters to regulate the rate of glucose absorption. Our in vitro data support this mechanism and also suggest that berry flavonoids may modulate post-prandial glycaemia by decreasing glucose transporter expression. Further studies are warranted to investigate the longer term effects of berry flavonoids on the management of glycaemia in human volunteers.

Delphinidin-3-glucoside (Dp) is a member of a family of bioactive compounds known as anthocyanins that occur naturally in pigmented plants and are known to ameliorate oxidative stress. Previous studies have showed that Dp decreased oxidative stress in vascular endothelial cells, however, the underlying mechanisms remain largely unknown. In the present study, we showed that pretreatment with Dp significantly suppressed oxidized low-density lipoprotein (oxLDL)-induced cell proliferation inhibition and apoptosis in primary human umbilical vein endothelial cells (HUVECs). Also, Dp pretreatment attenuated oxLDL-induced mitochondrial dysfunction via decreased reactive oxygen species (ROS) and superoxide anion generation, thereby repressing mitochondrial membrane potential and closing mitochondrial permeability transition pore. Furthermore, in vitro and in vivo data showed that Dp was transported into endothelial cells in a temperature, concentration, and time-dependent manner via the sodium-dependent glucose transporter (SGLT1). Suppression of SGLT1 by its substrate glucose, its inhibitor phlorizin or SGLT1 siRNA blocked Dp transportation. Repression of SGLT1 significantly inhibited Dp function of ameliorating mitochondrial dysfunction induced by pro-apoptotic factors (Apoptosis-inducing factor, Cytochrome c, Caspase-3 and Bax/Bcl-2 ratio). Taken together, our data indicate that Dp protects VECs via the SGLT1-ROS-mitochodria pathway. This new insight may help to elucidate the molecular mechanisms underlying the vascular protection afforded by Dp, and anthocyanins in general, in the context of prevention of endothelial dysfunction and atherosclerosis.

Sodium/glucose cotransporter 2 (SGLT2) inhibitors are oral hypoglycemic agents used to treat patients with diabetes mellitus. SGLT2 inhibitors block reabsorption of filtered glucose by inhibiting SGLT2, the primary glucose transporter in the proximal tubular cell (PTC), leading to glycosuria and lowering of serum glucose. We examined the renoprotective effects of the SGLT2 inhibitor empagliflozin to determine whether blocking glucose entry into the kidney PTCs reduced the inflammatory and fibrotic responses of the cell to high glucose. We used an in vitro model of human PTCs. HK2 cells (human kidney PTC line) were exposed to control 5 mM, high glucose (HG) 30 mM or the profibrotic cytokine transforming growth factor beta (TGFβ1; 0.5 ng/ml) in the presence and absence of empagliflozin for up to 72 h. SGLT1 and 2 expression and various inflammatory/fibrotic markers were assessed. A chromatin immunoprecipitation assay was used to determine the binding of phosphorylated smad3 to the promoter region of the SGLT2 gene. Our data showed that TGFβ1 but not HG increased SGLT2 expression and this occurred via phosphorylated smad3. HG induced expression of Toll-like receptor-4, increased nuclear deoxyribonucleic acid binding for nuclear factor kappa B (NF-κB) and activator protein 1, induced collagen IV expression as well as interleukin-6 secretion all of which were attenuated with empagliflozin. Empagliflozin did not reduce high mobility group box protein 1 induced NF-κB suggesting that its effect is specifically related to a reduction in glycotoxicity. SGLT1 and GLUT2 expression was not significantly altered with HG or empagliflozin. In conclusion, empagliflozin reduces HG induced inflammatory and fibrotic markers by blocking glucose transport and did not induce a compensatory increase in SGLT1/GLUT2 expression. Although HG itself does not regulate SGLT2 expression in our model, TGFβ increases SGLT2 expression through phosphorylated smad3.

MAP17 is a membrane-associated protein that is overexpressed in human tumors. Because the expression of MAP17 increases reactive oxygen species (ROS) generation through SGLT1 in cancer cells, in the present work, we investigated whether MAP17 and/or SGLT1 might be markers for the activity of treatments involving oxidative stress, such as cisplatin or radiotherapy. First, we confirmed transcriptional alterations in genes involved in the oxidative stress induced by MAP17 expression in HeLa cervical tumor cells and found that Hela cells expressing MAP17 were more sensitive to therapies that induce ROS than were parental cells. Furthermore, MAP17 increased glucose uptake through SGLT receptors. We then analyzed MAP17 and SGLT1 expression levels in cervical tumors treated with cisplatin plus radiotherapy and correlated the expression levels with patient survival. MAP17 and SGLT1 were expressed in approximately 70% and 50% of cervical tumors of different types, respectively, but they were not expressed in adenoma tumors. Furthermore, there was a significant correlation between MAP17 and SGLT1 expression levels. High levels of either MAP17 or SGLT1 correlated with improved patient survival after treatment. However, the patients with high levels of both MAP17 and SGLT1 survived through the end of this study. Therefore, the combination of high MAP17 and SGLT1 levels is a marker for good prognosis in patients with cervical tumors after cisplatin plus radiotherapy treatment. These results also suggest that the use of MAP17 and SGLT1 markers may identify patients who are likely to exhibit a better response to treatments that boost oxidative stress in other cancer types.

Background: Canagliflozin is a sodium glucose co-transporter (SGLT) 2 inhibitor in clinical development for the treatment of type 2 diabetes mellitus (T2DM)., Methods: (14)C-alpha-methylglucoside uptake in Chinese hamster ovary-K cells expressing human, rat, or mouse SGLT2 or SGLT1; (3)H-2-deoxy-d-glucose uptake in L6 myoblasts; and 2-electrode voltage clamp recording of oocytes expressing human SGLT3 were analyzed. Graded glucose infusions were performed to determine rate of urinary glucose excretion (UGE) at different blood glucose (BG) concentrations and the renal threshold for glucose excretion (RT(G)) in vehicle or canagliflozin-treated Zucker diabetic fatty (ZDF) rats. This study aimed to characterize the pharmacodynamic effects of canagliflozin in vitro and in preclinical models of T2DM and obesity., Results: Treatment with canagliflozin 1 mg/kg lowered RT(G) from 415±12 mg/dl to 94±10 mg/dl in ZDF rats while maintaining a threshold relationship between BG and UGE with virtually no UGE observed when BG was below RT(G). Canagliflozin dose-dependently decreased BG concentrations in db/db mice treated acutely. In ZDF rats treated for 4 weeks, canagliflozin decreased glycated hemoglobin (HbA1c) and improved measures of insulin secretion. In obese animal models, canagliflozin increased UGE and decreased BG, body weight gain, epididymal fat, liver weight, and the respiratory exchange ratio., Conclusions: Canagliflozin lowered RT(G) and increased UGE, improved glycemic control and beta-cell function in rodent models of T2DM, and reduced body weight gain in rodent models of obesity.

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase which promotes cell proliferation and survival, is abnormally overexpressed in numerous tumors of epithelial origin, including colorectal cancer (CRC). EGFR monoclonal antibodies have been shown to increase the median survival and are approved for the treatment of colorectal cancer. Histone deacetylases (HDACs), frequently overexpressed in colorectal cancer and several malignancies, are another attractive targets for cancer therapy. Several inhibitors of HDACs (HDACi) are developed and exhibit powerful antitumor abilities. In this study, human colorectal cancer cells treated with HDACi exhibited reduced EGFR expression, thereby disturbed EGF-induced ERK and Akt phosphorylation. HDACi also decreased the expression of SGLT1, an active glucose transporter found to be stabilized by EGFR, and suppressed the glucose uptake of cancer cells. HDACi suppressed the transcription of EGFR and class I HDACs were proved to be involved in this event. Chromatin immunoprecipitation analysis showed that HDACi caused the dissociation of SP1, HDAC3 and CBP from EGFR promoter. Our data suggested that HDACi could serve as a single agent to block both EGFR and HDAC, and may bring more benefits to the development of CRC therapy.