SGLT1 Activity Research Articles

Regulation of Enterocyte Brush Border Membrane Primary Na-Absorptive Transporters in Human Intestinal Organoid-Derived Monolayers.

In the small intestine, sodium (Na) absorption occurs primarily via two apical transporters, Na-hydrogen exchanger 3 (NHE3) and Na-glucose cotransporter 1 (SGLT1). The two primary Na-absorptive pathways were previously shown to compensatorily regulate each other in rabbit and rat intestinal epithelial cells. However, whether NHE3 and SGLT1 regulate one another in normal human enterocytes is unknown, mainly due to a lack of appropriate experimental models. To investigate this, we generated 2D enterocyte monolayers from human jejunal 3D organoids and used small interfering RNAs (siRNAs) to knock down NHE3 or SGLT1. Molecular and uptake studies were performed to determine the effects on NHE3 and SGLT1 expression and activity. Knockdown of NHE3 by siRNA in enterocyte monolayers was verified by qPCR and Western blot analysis and resulted in reduced NHE3 activity. However, in NHE3 siRNA-transfected cells, SGLT1 activity was significantly increased. siRNA knockdown of SGLT1 was confirmed by qPCR and Western blot analysis and resulted in reduced SGLT1 activity. However, in SGLT1 siRNA-transfected cells, NHE3 activity was significantly increased. These results demonstrate for the first time the functionality of siRNA in patient-derived organoid monolayers. Furthermore, they show that the two primary Na absorptive pathways in human enterocytes reciprocally regulate one another.

Comparison of SGLT1, SGLT2, and Dual Inhibitor Biological Activity in Treating Type 2 Diabetes Mellitus

Comparison of SGLT1, SGLT2, and Dual Inhibitor Biological Activity in Treating Type 2 Diabetes Mellitus

Sodium-glucose cotransporter 1 promotes the biofunctions of perivascular preadipocytes mediated by Akt/mTOR/p70S6K signaling pathway.

The influence of SGLT-1 on perivascular preadipocytes (PVPACs) and vascular remodeling is not well understood. This study aimed to elucidate the role and mechanism of SGLT-1-mediated PVPACs bioactivity. PVPACs were cultured in vitro and applied ex vivo to the carotid arteries of mice using a lentivirus-based thermosensitive in situ gel (TISG). The groups were treated with Lv-SGLT1 (lentiviral vector, overexpression), Lv-siSGLT1 (RNA interference, knockdown), or specific signaling pathway inhibitors. Assays were conducted to assess changes in cell proliferation, apoptosis, glucose uptake, adipogenic differentiation, and vascular remodeling in the PVPACs. Protein expression was analyzed by Western blotting, immunocytochemistry, and/or immunohistochemistry. The methyl thiazolyl tetrazolium (MTT) assay and Hoechst 33342 staining indicated that SGLT-1 overexpression significantly promoted PVPACs proliferation and inhibited apoptosis in vitro. Conversely, SGLT-1 knockdown exerted the opposite effect. Oil Red O staining revealed that SGLT-1 overexpression facilitated adipogenic differentiation, while its inhibition mitigated these effects. 3H-labeled glucose uptake experiments demonstrated that SGLT-1 overexpression enhanced glucose uptake by PVPACs, whereas RNA interference-mediated SGLT-1 inhibition had no significant effect on glucose uptake. Moreover, RT-qPCR, Western blotting, and immunofluorescence analyses revealed that SGLT-1 overexpression upregulated FABP4 and VEGF-A levels and activated the Akt/mTOR/p70S6K signaling pathway, whereas SGLT-1 knockdown produced the opposite effects. In vivo studies corroborated these findings and indicated that SGLT-1 overexpression facilitated carotid artery remodeling. Our study demonstrates that SGLT-1 activation of the Akt/mTOR/p70S6K signaling pathway promotes PVPACs proliferation, adipogenesis, glucose uptake, glucolipid metabolism, and vascular remodeling.NEW & NOTEWORTHY SGLT-1 is expressed in PVPACs and can affect preadipocyte glucolipid metabolism and vascular remodeling. SGLT-1 promotes the biofunctions of PVPACs mediated by Akt/mTOR/p70S6K signaling pathway. Compared with caudal vein or intraperitoneal injection, the external application of lentivirus-based thermal gel around the carotid artery is an innovative attempt at vascular remodeling model, it may effectively avoid the transfection of lentiviral vector into the whole body of mice and the adverse effect on experimental results.

Why have SGLT2 Inhibitors Failed to Achieve the Desired Success in COVID-19?

The SARS-CoV-2 virus emerged towards the end of 2019 and caused a major worldwide pandemic lasting at least 2 years, causing a disease called COVID-19. SARS-CoV-2 caused a severe infection with direct cellular toxicity, stimulation of cytokine release, increased oxidative stress, disruption of endothelial structure, and thromboinflammation, as well as angiotensin-converting enzyme 2 (ACE2) down-regulation-mediated renin-angiotensin system (RAS) activation. In addition to glucosuria and natriuresis, sodium-glucose transport protein 2 (SGLT2) inhibitors (SGLT2i) cause weight loss, a decrease in glucose levels with an insulin-independent mechanism, an increase in erythropoietin levels and erythropoiesis, an increase in autophagy and lysosomal degradation, Na+/H+-changer inhibition, prevention of ischemia/reperfusion injury, oxidative stress and they have many positive effects such as reducing inflammation and improving vascular function. There was great anticipation for SGLT2i in treating patients with diabetes with COVID-19, but current data suggest they are not very effective. Moreover, there has been great confusion in the literature about the effects of SGLT2i on COVID-19 patients with diabetes . Various factors, including increased SGLT1 activity, lack of angiotensin receptor blocker co-administration, the potential for ketoacidosis, kidney injury, and disruptions in fluid and electrolyte levels, may have hindered SGLT2i's effectiveness against COVID-19. In addition, the duration of use of SGLT2i and their impact on erythropoiesis, blood viscosity, cholesterol levels, and vitamin D levels may also have played a role in their failure to treat the virus. This article aims to uncover the reasons for the confusion in the literature and to unravel why SGLT2i failed to succeed in COVID-19 based on some solid evidence as well as speculative and personal perspectives.

Comparison of SGLT1, SGLT2, and Dual Inhibitor biological activity in treating Type 2 Diabetes Mellitus.

Diabetes Mellitus Type 2 (T2D) is an emerging health burden in the USand worldwide, impacting approximately 15% of Americans. Current front-line therapeutics for T2D patients include sulfonylureas that act to reduce A1C and/or fasting blood glucose levels, or Metformin that antagonizes the action of glucagon to reduce hepatic glucose production. Next generation glucomodulatory therapeutics target members of the high-affinity glucose transporter Sodium-Glucose-Linked-Transporter (SGLT) family. SGLT1 is primarily expressed in intestinal epithelium, whose inhibition reduces dietary glucose uptake, whilst SGLT2 is highly expressed in kidney - regulating glucose reabsorption. A number of SGLT2 inhibitors are FDA approved whilst SGLT1 and dual SGLT1 & 2 inhibitor are currently in clinical trials. Here, we discuss and compare SGLT2, SGLT1, and dual inhibitors' biochemical mechanism and physiological effects.

Increased intestinal permeability and downregulation of absorptive ion transporters Nhe3, Dra, and Sglt1 contribute to diarrhea during Clostridioides difficile infection

ABSTRACT Background & Aim Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhea and pseudomembranous colitis. Two protein toxins, TcdA and TcdB, produced by C. difficile are the major determinants of disease. However, the pathophysiological causes of diarrhea during CDI are not well understood. Here, we investigated the effects of C. difficile toxins on paracellular permeability and apical ion transporters in the context of an acute physiological infection. Methods We studied intestinal permeability and apical membrane transporters in female C57BL/6J mice. Üssing chambers were used to measure paracellular permeability and ion transporter function across the intestinal tract. Infected intestinal tissues were analyzed by immunofluorescence microscopy and RNA-sequencing to uncover mechanisms of transporter dysregulation. Results Intestinal permeability was increased through the size-selective leak pathway in vivo during acute CDI in a 2-day-post infection model. Chloride secretory activity was reduced in the cecum and distal colon during infection by decreased CaCC and CFTR function, respectively. SGLT1 activity was significantly reduced in the cecum and colon, accompanied by ablated SGLT1 expression in colonocytes and increased luminal glucose concentrations. SGLT1 and DRA expression was ablated by either TcdA or TcdB during acute infection, but NHE3 was decreased in a TcdB-dependent manner. The localization of key proteins that link filamentous actin to the ion transporters in the apical plasma membrane was unchanged. However, Sglt1, Nhe3, and Dra were drastically reduced at the transcript level, implicating downregulation of ion transporters in the mechanism of diarrhea during CDI. Conclusions CDI increases intestinal permeability and decreases apical abundance of NHE3, SGLT1, and DRA. This combination likely leads to dysfunctional water and solute absorption in the large bowel, causing osmotic diarrhea. These findings provide insights into the pathophysiological mechanisms underlying diarrhea and may open novel avenues for attenuating CDI-associated diarrhea.

Medium‐chain fatty acids inhibit jejunal SGLT1 activity in vitro and suppress glucose absorption in vivo in mice

Medium‐chain triglycerides (MCT) in diet improves glucose tolerance and insulin sensitivity, compared to long‐chain triglycerides (LCT). However, the direct effects of medium‐chain fatty acids (MCFA) on glucose transport have not been studied. Luminal glucose is absorbed in the small intestine through the electrogenic sodium‐dependent glucose transporter SGLT1 and electroneutral glucose transporter GLUT2. We thus examined the effects of luminal MCFA on electrogenic SGLT1 activity and glucose absorption in mice.SGLT1 activity was assessed in the Ussing chambered, muscle‐stripped jejunal mucosa by measuring phlorizin (Phz)‐sensitive short‐circuit current (Isc) evoked by luminal addition of glucose (10 mM). Sodium propionate (C3), caproate (C6), caprylate (C8), caprate (C10), laurate (C12) and myristate (C14) were luminally added before or after glucose application. Furthermore, the effects of C10 on glucose absorption were assessed by oral glucose tolerance test (OGTT, glucose 2g/kg body weight, ig) with or without C10 treatment (1.94g/kg, ig) in conscious mice. Moreover, the portal venous (PV) blood glucose levels were measured though the PV cannulation, followed by intraduodenal bolus injection of glucose solution (100 mM) with or without C10 (10 mM) in the anesthetized mice.Luminal pretreatment of C10 and C12 dose‐dependently inhibited the glucose‐induced Isc increase at IC50 = 1.02 mM and 1.06 mM, respectively. C8 also inhibited the glucose‐induced Isc increase, but less effective (IC50 8.4 mM), whereas C3, C6 and C14 had little effect. C10 (10 mM) also inhibited methyl α‐D‐glucopyranoside (αMG)‐induced Isc increase. Post‐treatment of C10 and C12 (10 mM) rapidly reduced the increased Isc by glucose, mimicking the inhibitory effect of Phz (1 mM). OGTT showed that glucose levels reached the peak at 30 min, while C10 pretreatment remarkably inhibited glucose increase. Furthermore, intraduodenal injection of glucose solution increased the PV glucose levels at 10 – 20 min, while the addition of C10 in the glucose solution abolished the response.These data suggest that C10 and C12 act as the SGLT1 inhibitor. Since MCT releases C10 and C12 via digestion by pancreatic lipase, slower than glucose release by maltase/sucrase, free C10/C12 solution may be more effective therapeutics for the treatment of type 2 diabetes.

Regulation of Glucose Insulinotropic Peptide and Intestinal Glucose Transporters in the Diet-Induced Obese Mouse.

Our recent studies have shown that glucose-dependent insulinotropic polypeptide (GIP), but not glucagon-like peptide 1 (GLP-1), augments Na-glucose transporter 1- (SGLT1-) mediated glucose absorption in mouse jejunum. Na-dependent glucose absorption sharply rose and peaked in 3 months of high-fat (i.e., obese) compared to normal (i.e., normal weight) diet fed animals. Previous studies have shown that GIP-augmented SGLT1 and PEPT1 (peptide transporter 1) are regulated by protein kinase A (PKA) signaling in mouse jejunum. Additional studies have indicated that cAMP and PI3 kinase signaling augment PEPT1 through EPAC and AKT activation pathways, respectively, through increased apical PEPT1 trafficking in intestinal epithelial cells. However, little is known about how the signaling glucose transport paradigm is altered over a long period. Early on, increased glucose absorption occurs through SGLT1, but as the obesity and diabetes progress, there is a dramatic shift towards a Na-independent mechanism. Surprisingly, at the peak of glucose absorption during the fifth month of the progression of obesity, the SGLT1 activity was severely depressed, while a Na-independent glucose absorptive process begins to appear. Since glucose transporter 2 (GLUT2) is expressed on the apical membrane of the small intestine in obese patients and animal models of obesity, it was hypothesized to be the new more efficient route. Western blot analyses and biotinylation of the apical membrane revealed that the GIP expression increases in the obese animals and its trafficking to the apical membrane increases with the GIP treatment.

Computational Modelling of Glucose Uptake by SGLT1 and Apical GLUT2 in the Enterocyte.

It has been suggested that glucose absorption in the small intestine depends on both constitutively expressed SGLT1 and translocated GLUT2 in the brush border membrane, especially in the presence of high levels of luminal glucose. Here, we present a computational model of non-isotonic glucose uptake by small intestinal epithelial cells. The model incorporates apical uptake via SGLT1 and GLUT2, basolateral efflux into the blood via GLUT2, and cellular volume changes in response to non-isotonic conditions. The dependence of glucose absorption on luminal glucose, blood flow rate, and inlet blood glucose concentration is studied. Uptake via apical GLUT2 is found to be sensitive to all these factors. Under a range of conditions, the maximum apical GLUT2 flux is about half of the SGLT1 flux and is achieved at high luminal glucose (> 50 mM), high blood flow rates, and low inlet blood concentrations. In contrast, SGLT1 flux is less sensitive to these factors. When luminal glucose concentration is less than 10 mM, apical GLUT2 serves as an efflux pathway for glucose to move from the blood to the lumen. The model results indicate that translocation of GLUT2 from the basolateral to the apical membrane increases glucose uptake into the cell; however, the reduction of efflux capacity results in a decrease in net absorption. Recruitment of GLUT2 from a cytosolic pool elicits a 10–20% increase in absorption for luminal glucose levels in the a 20–100 mM range. Increased SGLT1 activity also leads to a roughly 20% increase in absorption. A concomitant increase in blood supply results in a larger increase in absorption. Increases in apical glucose transporter activity help to minimise cell volume changes by reducing the osmotic gradient between the cell and the lumen.

IL-25 Treatment Improves Metabolic Syndrome in High-Fat Diet and Genetic Models of Obesity.

IntroductionEndemic obesity is considered the driving force for the dramatic increase in incidence of type 2 diabetes (T2D). There is mounting evidence that chronic, low-grade inflammation driven by Th1/Th17 cells and M1 macrophages, is a critical link between obesity and insulin resistance. IL-25 promotes development of a Th2 immune response and M2 macrophages that counteract the inflammation associated with obesity and T2D.MethodsMice were fed a high-fat diet (HFD) for 16 weeks and then treated with IL-25 or BSA as a control for 21 days. Body weight, blood glucose levels, intraperitoneal glucose tolerance, and gene expression were evaluated in mice treated with BSA or IL-25. Ob/ob mice fed a normal control diet were also treated with BSA or IL-25 and body weight and blood glucose levels were measured. Transepithelial electrical resistance and sodium-linked glucose absorption were determined in muscle-free small intestinal tissue and glucose absorption assessed in vitro in intestinal epithelial and skeletal muscle cell lines.ResultsAdministration of IL-25 to HFD fed mice reversed glucose intolerance, an effect mediated in part by reduction in SGLT-1 activity and Glut2 expression. Importantly, the improved glucose tolerance in HFD mice treated with IL-25 was maintained for several weeks post-treatment indicating long-term changes in glucose metabolism in obese mice. Glucose intolerance was also reversed by IL-25 treatment in genetically obese ob/ob mice without inducing weight loss. In vitro studies demonstrated that glucose absorption was inhibited by IL-25 treatment in the epithelial IPEC-1 cells but increased glucose absorption in the L6 skeletal muscle cells. This supports a direct cell-specific effect of IL-25 on glucose metabolism.ConclusionThese results suggest that the IL-25 pathway may be a useful target for the treatment of metabolic syndrome.

Dapagliflozin attenuates arrhythmic vulnerabilities by regulating connexin43 expression via the AMPK pathway in post-infarcted rat hearts

We have demonstrated that dapagliflozin, a sodium-glucose cotransporter (SGLT) 2 inhibitor, attenuates reactive oxygen species (ROS) production. Connexin43 playing a role in ventricular arrhythmia is sensitive to redox status. No data are available on the effects of dapagliflozin on arrhythmogenesis. This study was to determine whether dapagliflozin attenuated arrhythmias through modulating AMP-activated protein kinase (AMPK)/free radicals-induced connexin43 after myocardial infarction. After coronary ligation, normoglycemic male Wistar rats were randomized to either vehicle or dapagliflozin (0.1 mg/kg per day) for 4 weeks. Myocardial ROS levels were significantly increased (p < 0.05) and connexin43 levels were substantially decreased after myocardial infarction (p < 0.05). Dapagliflozin administration was associated with increased SGLT1, attenuated ROS and increased connexin43 levels in myocardium (all p < 0.05). During programmed electrical stimulation, arrhythmic severity was significantly improved in the dapagliflozin-treated infarcted rats than those in the vehicle-treated infarcted rats (p < 0.05). Dapagliflozin significantly increased AMPK phosphorylation compared to vehicle after infarction (p < 0.05). Inhibition of AMPK signaling by SBI-0206965 prevented increased SGLT1 and blocked the effects of dapagliflozin on attenuated ROS levels and increased connexin43 phosphorylation (all p < 0.05). SGLT1 inhibited by KGA-2727 showed attenuated ROS levels and increased connexin43 phosphorylation (both p < 0.05) although AMPK phosphorylation was not changed, implying SGLT1 activation was mediated by AMPK in dapagliflozin-treated hearts. Dapagliflozin-treated hearts had significantly increased connexin43 phosphorylation (p < 0.05), which was significantly decreased after adding 3-morpholinosydnonimine (p < 0.05). These data indicate that clinically-relevant dapagliflozin concentrations decreased free radicals content and increased connexin43 levels through AMPK-dependent and SGLT1-independent mechanisms, which attenuated ventricular arrhythmias in the normoglycemic infarcted rats.

19 DIET-INDUCED OBESITY UNIQUELY REGULATES GLUCOSE AND NACL ABSORPTION IN SPRAGUE DAWLEY RAT SMALL INTESTINAL EPITHELIAL CELLS

Background Traditional coupled NaCl absorption occurs via the dual operation of Na:H (NHE3) and Cl:HCO3 (DRA/PAT1) exchange in the brush border membrane of villus cells in the mammalian small intestine. In genetic models of obesity (Zucker rat and Tallyho mouse) and humans, coupled NaCl absorption was not affected. However, in these animal models, a novel coupling of Na-glucose co-transport (SGLT1) and Cl:HCO3 exchange was increased, potentially important in the pathogenesis of diabetes and hypertension associated with obesity. Human obesity is both genetic and environmental (e.g. diet mediated). But it is not known whether this novel coupled NaCl absorptive pathway is affected in diet-induced obesity (DIO). Hypothesis Novel coupled NaCl absorption mediated by SGLT1 and DRA/PAT1 is stimulated in DIO. Aim Determine the mechanisms of regulation of SGLT1 and DRA/PAT1 in DIO. Methods Sprague Dawley rats were fed with a high-fat diet (HFD; 60% fat calories) from 12-18 wks of age to induce obesity. Control rats were fed with standard chow. BBM vesicles (BBMV) were prepared from villus cells by Mg precipitation. Phlorizine sensitive, 3H-OMG uptake for SGLT1; amiloride-sensitive, 22Na uptake for NHE3 and DIDS sensitive 36Cl uptake for Cl:HCO3 exchange was performed in villus cell BBMV. Na/KATPase activity was determined as inorganic phosphate release. Western blot studies were performed using rat specific antibodies. Results In intact rat villus cells, SGLT1 uptake was significantly increased in HFD rats (2.4±0.2 nmol/mg protein×2 min in control and 6.4±0.6 in HFD; n=4, p Conclusions In DIO, BBM SGLT1 activity was stimulated in villus cells from HFD rats. This is not due to the altered Na extruding capacity of the cell. Cl:HCO3 exchange was also stimulated in DIO while NHE3 remained unaffected. These data indicate that in DIO, while the traditional coupled NaCl absorption is unaffected, a novel coupled NaCl absorptive pathway, via SGLT1 and DRA/PAT1 is stimulated resulting in enhanced glucose and NaCl absorption which is likely important in the pathophysiology of diabetes and hypertension of obesity.

Beta-Glucan From Barley Attenuates Post-prandial Glycemic Response by Inhibiting the Activities of Glucose Transporters but Not Intestinal Brush Border Enzymes and Amylolysis of Starch

Beta (β)-glucan (BG) from cereal grains is associated with lowering post-prandial blood glucose but the precise mechanism is not well-elucidated. The main aim of this study was to understand the mechanism through which BG from barley affects post-prandial glycemic response. Waffles containing 0, 1, 2, and 3 g barley BG and the same amount of available carbohydrate (15 g) were fed to the TIM-1 dynamic gastrointestinal digestion system to study the effect of BG on starch hydrolysis. Intestinal acetone powder and Xenopus laevis oocytes were used to study BG's effect on mammalian intestinal α-glucosidase and glucose transporters. The presence of BG did not significantly affect the in vitro starch digestion profiles of waffles suggesting that BG does not affect α-amylase activity. Intestinal α-glucosidase and glucose transport activities were significantly (p < 0.0001) inhibited in the presence of barley BG. Interestingly, BG viscosity did not influence α-amylase, α-glucosidase, GLUT2, and SGLT1 activities. This study provides the first evidence for the mechanism by which BG from barley attenuates post-prandial glycemic response is via alteration of α-glucosidase, GLUT2, and SGLT1 activity, but not amylolysis of starch. The decrease in post-prandial blood glucose in the presence of BG is likely a consequence of the interaction between BG and membrane active proteins (brush border enzymes and glucose transporters) as opposed to the commonly held hypothesis that increased viscosity caused by BG inhibits starch digestion.

Inducible Nitric Oxide Regulates Na-Glucose Co-transport in a Spontaneous SAMP1/YitFc Mouse Model of Chronic Ileitis.

In mammalian small intestine, glucose is primarily absorbed via Na-dependent glucose co-transporter (SGLT1) on the brush border membrane (BBM) of absorptive villus cells. Malabsorption of nutrients (e.g., glucose) leads to malnutrition, a common symptom of inflammatory bowel disease (IBD), where the mucosa is characterized by chronic inflammation. Inducible nitric oxide (iNO) is known to be elevated in IBD mucosa. SAMP1/YitFc (SAMP1) mouse is a spontaneous model of chronic ileitis that develops lesions in its terminal ileum, very similar to human IBD. How SGLT1 may be affected in SAMP1 model of chronic ileitis is unknown. Ten-week-old SAMP1 mice with AKR mice as control were treated with N6-(1-iminoethyl)-L-lysine dihydrochloride (L-NIL) to inhibit iNO production. Intracellular NO levels were found to be increased in villus cells from SAMP1 mice. Moreover, SGLT1 and Na+/K+-ATPase activities and BBM SGLT1 expression were significantly decreased. However, L-NIL treatment reduced the intracellular iNO production, and reversed both downregulated SGLT1 and Na+/K+-ATPase activities in SAMP1 mice. Inhibition of iNO by L-NIL treatment also significantly reversed the BBM SGLT1 protein expression in SAMP1 mice. L-NIL reversed the inflammation mediated downregulation of SGLT1 activity by restoring the BBM SGLT1 expression. Thus, regulation of SGLT1 in chronic ileitis is likely mediated by iNO.

Sleeve Gastrectomy Surgery Improves Glucose Metabolism by Downregulating the Intestinal Expression of Sodium–Glucose Cotransporter-3

Aims Sleeve gastrectomy (SG) has been proven effective in the treatment of obesity and type 2 diabetes. We hypothesized that SGLT3 may play an important role in the mechanism of glucose control and weight loss after SG. Materials and methods Daily body weight and food intake were measured in SG and sham-operated mice. Glucose tolerance test, SGLT3 agonist (αMG), and SGLT1 inhibitor (phlorizin) perfusion experiments were used to detect changes in intestinal SGLT3 and SGLT1 activity following SG. Expression of SGLT3a and SGLT1 was assessed at 2 weeks, 1 month after surgery by quantitative PCR and fluorescence immunoassay. Hematoxylin and eosin staining was used to detect morphological changes in the villi. SGLT3 and SGLT1 expression was measured after stimulation of human intestinal epithelial cells (HIEC). Results Both the body weight and daily food intake of the SG-treated mice decreased within 30 days after surgery. Oral glucose absorption was significantly reduced at 30 days. The intestinal stimulation proved that SG can improve glucose metabolism, which can be reversed by αMG and enhanced by phlorizin. Villus height and surface area of the intestine in SG mice decreased after surgery. mRNA expression of SGLT3a and SGLT1 decreased at 2 weeks and 1 month after SG, immunofluorescence also confirmed these changes. HIEC stimulation confirmed that αMG could increase the expression of SGLT3 and SGLT1, but the expression of SGLT1 was down regulated when phlorizin was added to the medium. Conclusion The results suggest that reducing SGLT3 expression might contribute to lowering blood glucose and controlling body weight after SG.

Lysophosphatidic Acid Increases Maturation of Brush Borders and SGLT1 Activity in MYO5B-deficient Mice, a Model of Microvillus Inclusion Disease

Myosin VB (MYO5B) is an essential trafficking protein for membrane recycling in gastrointestinal epithelial cells. The inactivating mutations of MYO5B cause the congenital diarrheal disease, microvillus inclusion disease (MVID). MYO5B deficiency in mice causes mislocalization of SGLT1 and NHE3, but retained apical function of CFTR, resulting in malabsorption and secretory diarrhea. Activation of lysophosphatidic acid (LPA) receptors can improve diarrhea, but the effect of LPA on MVID symptoms is unclear. We investigated whether LPA administration can reduce the epithelial deficits in MYO5B-knockout mice. Studies were conducted with tamoxifen-induced, intestine-specific knockout of MYO5B (VilCreERT2;Myo5bflox/flox) and littermate controls. Mice were given LPA, an LPAR2 agonist (GRI977143), or vehicle for 4 days after a single injection of tamoxifen. Apical SGLT1 and CFTR activities were measured in Üssing chambers. Intestinal tissues were collected, and localization of membrane transporters was evaluated by immunofluorescence analysis in tissue sections and enteroids. RNA sequencing and enrichment analysis were performed with isolated jejunal epithelial cells. Daily administration of LPA reduced villus blunting, frequency of multivesicular bodies, and levels of cathepsins in intestinal tissues of MYO5B-knockout mice compared with vehicle administration. LPA partially restored the brush borderheight and the localization of SGLT1 and NHE3 in smallintestine of MYO5B-knockout mice and enteroids. The SGLT1-dependent short-circuit current was increased and abnormalCFTR activities were decreased in jejunum from MYO5B-knockout mice given LPA compared with vehicle. LPA may regulate a MYO5B-independent trafficking mechanism and brush border maturation, and therefore be developed for treatment of MVID.

Glucose transport in lymphocytes.

Glucose uptake into lymphocytes is accomplished by non-concentrative glucose carriers of the GLUT family (GLUT1, GLUT3, GLUT4, GLUT6) and/or by the Na+-coupled glucose carrier SGLT1. The latter accumulates glucose against glucose gradients and is still effective at very low extracellular glucose concentrations. Signaling involved in SGLT1 expression and activity includes protein kinase A (PKA), protein kinase C (PKC), serum- and glucocorticoid-inducible kinase (SGK1), AMP-activated kinase (AMPK), and Janus kinases (JAK2 and JAK3). Glucose taken up is partially stored as glycogen. In hypoxic environments, such as in tumors as well as infected and inflamed tissues, lymphocytes depend on energy production from glycogen-dependent glycolysis. The lack of SGLT1 may compromise glycogen storage and thus lymphocyte survival and function in hypoxic tissues. Accordingly, in mice, genetic knockout of sglt1 compromised bacterial clearance following Listeria monocytogenes infection leading to an invariably lethal course of the disease. Whether the effect was due to the lack of sglt1 in lymphocytes or in other cell types still remains to be determined. Clearly, additional experimental effort is required to define the role of glucose transport by GLUTs and particularly by SGLT1 for lymphocyte survival and function, as well as orchestration of the host defense against tumors and bacterial infections.

Effectiveness in the inhibition of dapagliflozin and canagliflozin on M-type K+ current and α-methylglucoside-induced current in pituitary tumor (GH3) and pheochromocytoma PC12 cells

Dapagliflozin (DAPA) or canagliflozin (CANA), Na+-dependent glucose co-transporter type 2 (SGLT2) inhibitors, were used for treatment of type II diabetes mellitus. Addition of DAPA or CANA suppressed M-type K+ current (IK(M)) in pituitary tumor (GH3) and pheochromocytoma PC12 cells. The IC50 value for DAPA- or CANA-mediated inhibition of IK(M) in GH3 cells was 0.11 or 0.42 μM, respectively. The presence of DAPA (0.1 μM) shifted the steady-state activation of IK(M) to less depolarized potential without changing the gating charge of the current. During high-frequency depolarizing pulses, IK(M) magnitude was reduced by DAPA; however, DAPA-induced block of IK(M) remained effective. The amplitude of neither erg-mediated K+ current nor hyperpolarization-activated cation current in GH3 cells was modified in the presence of 1 μM DAPA. Alternatively, addition of DAPA, CANA, phlorizin or chlorotoxin effectively suppressed α-methylglucoside-(αMG-) induced current (IαMG) in GH3 cells, albeit inability of tefluthrin (activator of INa) to suppress this current. DAPA shifted the charge-voltage relation of presteady-state IαMG in a rightward and downward direction with no change in the gating charge of the IαMG. Under current-clamp recordings, subsequent additions of DAPA, but still in the continued presence of αMG, increased the firing rate of spontaneous action potentials stimulated by αMG. Our results suggested that activity of SGLT was expressed functionally in GH3 and PC12 cells. Therefore, inhibitory actions of DAPA or CANA on the amplitude and gating of IK(M) might provide a yet unidentified mechanism through which the SGLT1 or SGLT2 activity were attenuated in unclamped cells occurring in vivo.

LEFTY2/endometrialbleeding-associated factor up-regulates Na+Coupled Glucose Transporter SGLT1 expression and Glycogen Accumulation in Endometrial Cancer Cells.

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.

Moderate Alcohol Consumption Uniquely Regulates Sodium-Dependent Glucose Co-Transport in Rat Intestinal Epithelial Cells In Vitro and In Vivo

Chronic alcohol use often leads to malnutrition. However, how the intestinal absorption of nutrients such as glucose may be affected during moderate ethanol use has not been investigated. Glucose is absorbed via sodium (Na)-dependent glucose co-transport (SGLT1; SLC5A1) along the brush border membrane (BBM) of intestinal absorptive villus cells. The aim of this study was to investigate how moderate alcohol consumption affects the absorption of glucose via SGLT1. Intestinal epithelial cells (IEC-18; rat) were exposed to 8.64mM ethanol over 1, 3, 6, and 12 h. Rats (16-wk-old, male, Sprague-Dawley) were administered 2g/kg ethanol over 1, 3, and 6 h. Na-dependent 3H-O-methyl-d-glucose uptake was measured to assess SGLT1 activity. Na-K-ATPase activity was measured as a function of inorganic phosphate release. Protein expression was analyzed by Western blot analysis and immunohistochemical staining. Ethanol significantly decreased Na-dependent glucose absorption in enterocytes in vitro (ethanol treatment: 48.4% of controls at 1 h; P<0.01) and in vivo (ethanol treatment: 60.0% of controls at 1 h; P<0.01). Na-K-ATPase activity was significantly inhibited in vitro (ethanol treatment: 36.9% of controls at 1 h; P<0.01) and in vivo (ethanol treatment: 42.1% of controls at 1 h; P<0.01). Kinetic studies showed that the mechanism of inhibition of Na-glucose co-transport was secondary to a decrease in the affinity (1/Km) of the co-transporter for glucose both in vitro and in vivo. Western blots and immunohistochemistry further demonstrated unaltered amounts of SGLT1 after ethanol treatment. Moderate ethanol significantly decreases glucose absorption in IEC-18 cells and in villus cells of Sprague-Dawley rats. The inhibition of SGLT1 is secondary to an altered Na gradient at the cellular level and secondary to diminished affinity of the co-transporter for glucose at the protein level in the BBM. These observations may, at least in part, explain 1 possible mechanism of the onset of malnutrition associated with alcohol consumption.