SGLT1 Gene Expression Research Articles

Oral administration of Bifidobacterium bifidum G9-1 alleviates rotavirus gastroenteritis through regulation of intestinal homeostasis by inducing mucosal protective factors.

Human rotavirus (RV) infection is a leading cause of dehydrating diarrhea in infants and young children worldwide. Since therapeutic approaches to RV gastroenteritis are limited to alleviation of dehydration with oral rehydration solutions, more direct approaches to palliate symptoms of RV gastroenteritis are required. Treatments with probiotics have been increasingly recognized as alternative safe and low cost treatments for moderate infectious diarrhea. In this study, Bifidobacterium bifidum G9-1 (BBG9-1), which has been used as an intestinal drug for several decades, was shown to have a remarkable protective effect against RV gastroenteritis in a suckling mice model. As well as prophylactic oral administration of BBG9-1 from 2 days before RV infection, therapeutic oral administration of BBG9-1 from 1 day after RV infection significantly alleviated RV-induced diarrhea. Therapeutic administration of BBG9-1 reduced various types of damage in the small intestine, such as epithelial vacuolization and villous shortening, and significantly diminished the infectious RV titer in mixtures of cecal contents and feces. It was also shown that therapeutic administration of BBG9-1 significantly increased the number of acidic mucin-positive goblet cells and the gene expression of mucosal protective factors including MUC2, MUC3, MUC4, TGFβ1 and TFF3 in the small intestine. This led to alleviation of low gut permeability shown as decreased gene expression levels of occludin, claudin-1 and villin-1 after RV infection. Furthermore, in the small intestine, therapeutic administration of BBG9-1 significantly palliated the decreased gene expression of SGLT-1, which plays an important role in water absorption. In the large intestine, administered BBG9-1 was shown to replicate to assimilate undigested nutrients, resulting in normalization of the abnormally high osmotic pressure. These results suggested that water malabsorption caused by RV infection was alleviated in mice administered BBG9-1. Thus, the present study showed that oral administration of BBG9-1 palliated diarrhea partly through protection against RV-induced lesions by inducing mucosal protective factors. Oral administration of BBG9-1 is thought to be an efficient method for management of an RV epidemic for both prophylactic and therapeutic purposes.

An Essential Role of cAMP Response Element Binding Protein in Ginsenoside Rg1-Mediated Inhibition of Na+/Glucose Cotransporter 1 Gene Expression.

The Na(+)/glucose cotransporter 1 (SGLT1) is responsible for glucose uptake in intestinal epithelial cells. It has been shown that the intestinal SGLT1 level is significantly increased in diabetic individuals and positively correlated with the pathogenesis of diabetes. The development of targeted therapeutics that can reduce the intestinal SGLT1 expression level is, therefore, important. In this study, we showed that ginsenoside Rg1 effectively decreased intestinal glucose uptake through inhibition of SGLT1 gene expression in vivo and in vitro. Transient transfection analysis of the SGLT1 promoter revealed an essential cAMP response element (CRE) that confers the Rg1-mediated inhibition of SGLT1 gene expression. Chromatin immunoprecipitation assay and targeted CRE-binding protein (CREB) silencing demonstrated that Rg1 reduced the promoter binding of CREB and CREB binding protein associated with an inactivated chromatin status. In addition, further studies showed that the epidermal growth factor receptor (EGFR) signaling pathway also plays an essential role in the inhibitory effect of Rg1; taken together, our study demonstrates the involvement of the EGFR-CREB signaling pathway in the Rg1-mediated downregulation of SGLT1 expression, which offers a potential strategy in the development of antihyperglycemic and antidiabetic treatments.

A gut microbial metabolite of ginsenosides, compound K, induces intestinal glucose absorption and Na(+) /glucose cotransporter 1 gene expression through activation of cAMP response element binding protein.

The Na(+) /glucose cotransporter 1 (SGLT1) plays a crucial role in glucose uptake in intestinal epithelial cells (IECs), which has been shown essential in ameliorating intestinal inflammation. Ginseng has historically been used to treat inflammatory disorders. Understanding the regulatory mechanism of ginseng-mediated induction of SGLT1 gene expression in human intestinal cells is therefore important. We demonstrate that ginsenoside compound K (CK) enhances SGLT1-mediated glucose uptake in mice and human intestinal Caco-2 cells. Transient transfection analysis using SGLT1 promoter-luciferase reporters demonstrated that the presence of an essential cAMP response element (CRE) is required for CK-mediated induction of SGLT1 gene expression. The ChIP assays indicated that increased CRE-binding protein (CREB) and CREB-binding protein (CBP) binding to the SGLT1 promoter in CK-treated cells is associated with an activated chromatin state. Our result showed that the increased CREB phosphorylation is directly correlated with SGLT1 expression in IECs. Further studies indicated that the epidermal growth factor receptor (EGFR) signaling pathway is involved in the CK-mediated effect. These findings provide a novel mechanism for the CK-mediated upregulation of SGLT1 expression through EGFR-CREB signaling activation, which could contribute to reducing gut inflammation.

Induction of histone H3K4 methylation at the promoter, enhancer, and transcribed regions of the Si and Sglt1 genes in rat jejunum in response to a high-starch/low-fat diet

ObjectiveHistone methylation patterns are associated with various aspects of biology, including transcriptional regulation. Methylation of histone H3 at lysine 4 (H3K4) leads to transcriptional activation through recruitment of transcription activation complexes onto target genes; in contrast, methylation of histone H3K9, or histone H4K20, leads to transcriptional inactivation attracting heterochromatin protein 1 (HP1). It is not yet known whether jejunal induction of sucrase-isomaltase (Si) and sodium-dependent glucose cotransporter (Sglt1) genes by intake of a high-starch/low-fat diet in rats is regulated by coordinated changes of these histone methylation events. In the present study, we investigated whether these histone modifications at the promoter, enhancer, and transcribed regions of Si and Sglt1 genes in rat jejunum are affected by consumption of a high-starch/low-fat diet. MethodsChromatin immunoprecipitation assays using antibodies against methylated-histone H3K4, H3K9, H4K20, and HP1 were performed at various regions associated with the Si and Sglt1 genes in jejunum of rats fed a high-starch/low-fat diet or a low-starch/high-fat diet for 7 d. ResultsFeeding rats the high-starch/low-fat diet induced mono-, di-, and trimethylation of histone H3K4 on the promoter and transcribed regions of the Si and Sglt1 genes. In contrast, methylation of histones H3K9 and H4K20, and binding of HP1 at these gene regions, were not affected by the high-starch/low-fat diet. ConclusionThese observations suggest that induction of Si and Sglt1 gene expression in rat jejunum by a high-starch/low-fat diet intake is positively associated with histone H3K4 methylation, but not with histone H3K9/H4K20 methylation, or with binding of HP1.

Development of a cell-based nonradioactive glucose uptake assay system for SGLT1 and SGLT2

Sodium-dependent glucose cotransporters (SGLT1 and SGLT2), which have a key role in the absorption of glucose in the kidney and/or gastrointestinal tract, have been proposed as a novel therapeutic strategy for diabetes and cardiomyopathy. Here we developed a simple cell-based, nonradioactive method for functional screening of SGLT1 and SGLT2 inhibitors. Stable cell lines expressing human SGLT1 and SGLT2 were established by transfecting HEK293 cells with vectors (pCMV6-Neo) having full-length human SGLT1 and SGLT2 and selecting the positive clones following neomycin treatment. We confirmed the gene expression of SGLT1 and SGLT2 by reverse transcription polymerase chain reaction (RT–PCR) and immunoblotting. Furthermore, to analyze the function of SGLTs, we incubated stable cell lines with 2-deoxyglucose or fluorescent d-glucose analog (2-NBDG) and performed glucose uptake assay. A significant (P<0.001) increase in glucose uptake was observed in both cell lines. The increased glucose uptake in both cell lines was completely inhibited when treated with nonspecific SGLT1/SGLT2 inhibitors and phlorizin (100μM), but not when treated with nonspecific sodium-independent facilitative glucose transporter (GLUT) inhibitors (100μM). Taken together, our data suggest that cell-based methods developed for screening SGLT1/SGLT2 inhibitors are phlorizin sensitive and specific for respective glucose transporters. This assay provides a simple and rapid method for identifying novel and selective SGLT inhibitors.

Transcript Profiles of Hexose Transporters in the Equine Gastrointestinal Tract

To test the hypothesis that hexose transporter transcripts are in lower abundance in the large compared with the small intestine of the horse, mRNA abundance of genes encoding for hexose transporters was quantified in small and large intestinal segments of the horse. Four adult horses were euthanized, and mucosal samples were taken from two segments of the small intestine (distal jejunum [DJ] and ileum [IL]) and three segments of the large intestine (cecum [CE], left ventral colon [LVC], and left dorsal colon [LDC]). Transcript abundance of candidate hexose transporter genes was determined using reverse transcription quantitative polymerase chain reaction. Primers for genes SLC2A1, SLC2A2, SLC2A5, and SLC5A1, encoding for the hexose transporter proteins GLUT1, GLUT2, GLUT5, and SGLT1, respectively, were designed using gene sequences from GenBank. The 2ΔΔCT method was used to compare mRNA abundance of candidate genes in the IL, CE, LVC, and LDC with that of the DJ. Relative to the DJ, mRNA abundance of GLUT1 was not different in the IL, CE, and LVC, and increased (P ≤ .05) in the LDC. GLUT2, GLUT5, and SGLT1 gene expression was not different in the IL, and decreased (P ≤ .01) in the CE, LVC, and LDC, relative to the DJ. The decrease in expression for the majority of the hexose transporter genes in the large intestine indicates that it has less capacity for glucose, galactose, or fructose absorption compared with the small intestine of the horse.

Expression of sglt1 gene in Cyprinus carpio and preparation of its polyclonal antibody

The high-affinity Na+/glucose contransporter Sglt1 is one of the important members of the sodium:solute symporter family(SSF),belonging to the homologous family 5(SLC5).The Sglt1 plays an important role in accumulating glucoses from intestinal or kidney epithelial cells against an adverse concentration gradient and maintaining the adjustment of metabolism.So far,few studies on Na+/glucose cotransporter have been reported in the freshwater fishes.This research would develop special antibodies of Sglt1,which could supply the foundation for the research of glucose metabolism in Cyprinus carpio intestines on molecular level.To study the molecular mechanism of glucose metabolism in freshwater fishes,the full-length cDNA of sglt1 in C.carpio with an ORF of 1 977 bp was cloned and the antigenicity of Sglt1 was first predicted.92(544-637)amino acids with strong antigenicity and immunogenicity were selected as target section of sglt1 in C.carpio and were cloned into plasmid pET-32a(+)vector which had restriction sites for EcoRⅠ and HindⅢ.The rcombinant plasmid named pET-32a(+)-sglt1-P was transformed into E.coli Rosetta.The Sglt1-P fusion protein of approximately 30 ku was highly expressed in E.coli Rosetta after being induced with IPTG.The purified fusion protein was used as antigen to immunize New Zealand Rabbits with ear margin veins by subcutaneous injection with power.The results of ELISA showed that the titer of the antiserum was about 1∶ 105.The Sglt1-P polyclonal antibody was used to determine the expression of Sglt1 protein in C.carpio intestines through immunohistochemical method.The result revealed that the antibody performed high affinity and specificity and could be applied to study the expression and locating of Sglt1 in the C.carpio.The Sglt1-P polyclonal antibody also could serve as an important research tool to study Sglt1 expression and transshipment activity of C.carpio.Meanwhile,the Sglt1-P polyclonal antibody could also be used to exploratively study the expression location and quantity of the Sglt1 transporters in other fishes.

Relative Contributions of Afferent Vagal Fibers to Resistance to Diet-Induced Obesity

We previously demonstrated vagal neural pathways, specifically subdiaphragmatic afferent fibers, regulate expression of the intestinal sodium-glucose cotransporter SGLT1, the intestinal transporter responsible for absorption of dietary glucose. We hypothesized targeting this pathway could be a novel therapy for obesity. We therefore tested the impact of disrupting vagal signaling by total vagotomy or selective vagal de-afferentation on weight gain and fat content in diet-induced obese rats. Male Sprague-Dawley rats (n=5-8) underwent truncal vagotomy, selective vagal de-afferentation with capsaicin, or sham procedure. Animals were maintained for 11 months on a high-caloric Western diet. Abdominal visceral fat content was assessed by magnetic resonance imaging together with weight of fat pads at harvest. Glucose homeostasis was assessed by fasting blood glucose and HbA1C. Jejunal SGLT1 gene expression was assessed by qPCR and immunoblotting and function by glucose uptake in everted jejunal sleeves. At 11-months, vagotomized rats weighed 19% less (P=0.003) and de-afferented rats 7% less (P=0.19) than shams. Vagotomized and de-afferented animals had 52% (P<0.0001) and 18% reduction (P=0.039) in visceral abdominal fat, respectively. There were no changes in blood glucose or glycemic indexes. SGLT1 mRNA, protein and function were unchanged across all cohorts at 11-months postoperatively. Truncal vagotomy led to significant reductions in both diet-induced weight gain and visceral abdominal fat deposition. Vagal de-afferentation led to a more modest, but clinically and statistically significant, reduction in visceral abdominal fat. As increased visceral abdominal fat is associated with excess morbidity and mortality, vagal de-afferentation may be a useful adjunct in bariatric surgery.

Pituitary Pars Intermedia Dysfunction Down-regulates mRNA Abundance of Genes Encoding GLUT4 and Insulin Receptor in the Small Intestinal Mucosa of the Horse

To test the hypothesis that hexose transporter transcripts are in lower abundance in the large compared with the small intestine of the horse, mRNA abundance of genes encoding for hexose transporters was quantified in small and large intestinal segments of the horse. Four adult horses were euthanized, and mucosal samples were taken from two segments of the small intestine (distal jejunum [DJ] and ileum [IL]) and three segments of the large intestine (cecum [CE], left ventral colon [LVC], and left dorsal colon [LDC]). Transcript abundance of candidate hexose transporter genes was determined using reverse transcription quantitative polymerase chain reaction. Primers for genes SLC2A1, SLC2A2, SLC2A5, and SLC5A1, encoding for the hexose transporter proteins GLUT1, GLUT2, GLUT5, and SGLT1, respectively, were designed using gene sequences from GenBank. The 2ΔΔCT method was used to compare mRNA abundance of candidate genes in the IL, CE, LVC, and LDC with that of the DJ. Relative to the DJ, mRNA abundance of GLUT1 was not different in the IL, CE, and LVC, and increased (P ≤ .05) in the LDC. GLUT2, GLUT5, and SGLT1 gene expression was not different in the IL, and decreased (P ≤ .01) in the CE, LVC, and LDC, relative to the DJ. The decrease in expression for the majority of the hexose transporter genes in the large intestine indicates that it has less capacity for glucose, galactose, or fructose absorption compared with the small intestine of the horse.

Histomorphology and small intestinal sodium-dependent glucose transporter 1 gene expression in piglets fed phytic acid and phytase-supplemented diets1

An experiment was conducted to determine the effect of dietary phytic acid (PA) and phytase supplementation on small intestinal histomorphology and Na-dependent glucose transporter 1 (SGLT1) gene expression in piglets. Twenty-four piglets with an average initial BW of 7.60 ± 0.73 kg were randomly assigned to 3 experimental diets, to give 8 piglets per diet. The diets were a casein-cornstarch-based diet that was supplemented with 0 or 2% PA, or 2% PA (as Na phytate) plus an Escherichia coli-derived phytase at 500 phytase units/kg. The basal diet was formulated to meet the 1998 NRC energy, digestible AA, mineral, and vitamin requirements for piglets. After 10 d of feeding, the piglets were killed to determine small intestinal histomorphology and small intestinal SGLT1 gene expression. Phytic acid supplementation did not affect (P > 0.1) villus height (VH) and the VH-to-crypt depth (CD) ratio, but did decrease (P < 0.05) CD in the jejunum. Phytase supplementation did not affect (P > 0.1) VH, CD, and the VH-to-CD ratio. Phytic acid supplementation reduced SGLT1 gene expression in the duodenum, jejunum, and ileum by 1.1-, 5.4-, and 2.4-fold, respectively. Phytase supplementation increased SGLT1 gene expression in the jejunum by 2.6-fold, but reduced SGLT1 gene expression in the duodenum and ileum by 2.0- and 4.0-fold, respectively. In conclusion, PA reduced CD in the jejunum and SGLT1 gene expression in the duodenum, jejunum, and ileum, whereas phytase supplementation increased the expression of SGLT1 in the jejunum. The reduced SGLT1 gene expression by PA implies that PA reduces nutrient utilization in pigs partly through reduced expression of SGLT1, which is involved in glucose and Na absorption. The increased expression of SGLT1 in the jejunum by phytase supplementation implies that phytase alleviated the negative effects of PA partly through increased expression of SGLT1.

Clock genes regulate circadian rhythmical change of sglt1 gene expression according to feeding schedule through binding of BMAL1 on the promoter region

Sodium‐D‐glucose cotransporter SGLT1 plays an important role in the absorption of glucose in the small intestine. To examine whether the circadian rhythmical expression of the intestinal sglt1 gene is regulated by clock genes, we determined the levels of mRNA and BMAL1 binding on the promoter regions of the sglt1 gene by real‐time RT‐PCR and chromatin immunoprecipitation assay, respectively, in the jejunum of mice receiving free access to diet and those with restricted feeding from 9:00 to 17:00 under light (7:00‐19:00)‐dark (19:00‐7:00) cycle. Jejunal expression of sglt1 gene gradually increased from 7:00, and reached a maximal level at 19:00 in mice with free access to diet. The peak was shifted to 7:00 in mice with restricted feeding schedule. The gene expression of bmal1, a transcription factor driving the central feedback loop of clock genes, displayed a maximal expression at the period when sglt1 gene expression started to increase. The gene expression of per1‐3, the clock genes coding negative regulators of BMAL1, was highest at or soon after the time of maximal expression of sglt1 gene in both groups. The binding of BMAL1 to the promoters of sglt1 and per2 genes was associated with their expressional changes. The results suggest that the circadian expressional change of sglt1 gene in the jejunum of mice which is cued by the feeding schedule is directly regulated by a feedback loop of clock genes.

The Effects of Fasting and Grazing on Na-glucose Cotransporter-1 (SGLT-1) Gene Expression of Rectal Epithelia in Beef Cattle

The expression of SGLT-1 mRNA has been reported in the small intestine of mammals and the rectum of chickens. However, the expression and functional significance of SGLT-1 in bovine rectum is not known. In this study, we studied the effects of fasting and grazing on SGLT-1 gene expression in biopsy epithelial tissue of bovine rectum. In Japanese Black beef cattle, i) SGLT-1 gene expression was measured by quantitative real-time PCR in the biopsy rectal epithelia samples obtained through an endoscope, ii) SGLT-1 gene expression in the rectal epithelial tissues increased at 48 and 72 h after fasting correlating with a decrease in body weight. iii) SGLT-1 gene expression decreased after one month from the start of grazing (May to June) and then stabilized until the end of the grazing period (June to October) in the rectal epithelial tissues of grazing cattle. In conclusion, it is clear that SGLT-1 gene expression in the rectal epithelial tissue is increased by a restricted dietary condition.

Gene Expression of Nutrient Transporters in the Small Intestine of Chickens from Lines Divergently Selected for High or Low Juvenile Body Weight

Nutrient transporters in the small intestine are responsible for dietary nutrient assimilation; therefore, the expression of these transporters can influence overall nutrient status as well as the growth and development of the animal. This study examined correlated responses to selection in the developmental gene expression of PepT1, EAAT3, SGLT1, and GLUT5 in the small intestine of chickens from lines divergently selected for 48 generations for high (HH) or low (LL) 56-d BW and their reciprocal crosses (HL and LH). Duodenum, jejunum, and ileum were collected from male and female chicks on embryonic d 20, day of hatch with no access to feed, and d 3, 7, and 14 posthatch. Total RNA was extracted, and nutrient transporter expression was assayed by real-time PCR using the relative quantification method. In comparing male and female HH and LL chicks, there was a mating combination × age × sex interaction for PepT1 expression (P < 0.001), a main effect of sex for EAAT3 (P < 0.05) and SGLT1 (P < 0.001) expression, and an age × sex interaction for SGLT1 expression (P < 0.001). These results demonstrate a sexual dimorphism in the capacity to absorb nutrients from the intestine, which has implications for the poultry industry with regard to diet formulations for straight-run and sex-separate grow-out operations. Results from comparing male LL, LH, HL, and HH chicks indicate that selection for high or low juvenile BW may have influenced the gene expression profiles of these nutrient transporters in the small intestine, which may contribute to the overall differences in the growth and development of these lines of chickens.

Inductions of histone H3 acetylation at lysine 9 on SGLT1 gene and its expression by feeding mice a high carbohydrate/fat ratio diet

Objective We examined in this study whether histone H3 acetylations on jejunal NA +/glucose transport-1 (SGLT1) gene are associated with induction of its gene by feeding mice a high carbohydrate/fat ratio diet. Methods The chromatin immunoprecipitation (ChIP) assays using antibodies of acetylated histone H3 were performed in jejunum of mice fed a high carbohydrate/fat ratio diet or a low carbohydrate/fat ratio for 7 d. Results The acetylations of histone H3 of lysine 9/14 were highly detected on the SGLT1 gene and the levels were enhanced by feeding mice a high carbohydrate/fat ratio diet. Histone H3 acetylation at lysine 9 was enhanced by feeding a high carbohydrate/fat ratio diet, whereas that at lysine 14 was not enhanced significantly. Conclusion These observations indicate that induction of SGLT1 gene expression by feeding a high carbohydrate/fat ratio diet is associated with acetylation of histone H3 at lysine 9 on the SGLT1 gene.

Yerba Maté (Ilex paraguariensis) Aqueous Extract Decreases Intestinal SGLT1 Gene Expression but Does Not Affect Other Biochemical Parameters in Alloxan-Diabetic Wistar Rats

Yerba maté (Ilex paraguariensis) is rich in polyphenols, especially chlorogenic acids. Evidence suggests that dietary polyphenols could play a role in glucose absorption and metabolism. The aim of this study was to evaluate the antidiabetic properties of yerba maté extract in alloxan-induced diabetic Wistar rats. Animals (n = 41) were divided in four groups: nondiabetic control (NDC, n = 10), nondiabetic yerba maté (NDY, n = 10), diabetic control (DC, n = 11), and diabetic yerba maté (DY, n = 10). The intervention consisted in the administration of yerba maté extract in a 1 g extract/kg body weight dose for 28 days; controls received saline solution only. There were no significant differences in serum glucose, insulin, and hepatic glucose-6-phosphatase activity between the groups that ingested yerba maté extract (NDY and DY) and the controls (NDC and DC). However, the intestinal SGLT1 gene expression was significantly lower in animals that received yerba maté both in upper (p = 0.007) and middle (p < 0.001) small intestine. These results indicate that bioactive compounds present in yerba maté might be capable of interfering in glucose absorption, by decreasing SGLT1 expression.

Expression of the sodium‐glucose cotransporter SGLT1 gene along the jejunal crypt‐villus axis measured by quantitative real time RT‐PCR in the formula‐fed neonatal pig

This study was conducted to quantify the SGLT1 mRNA expression along the jejunal crypt-villus axis in fed neonatal pigs. Six Yorkshire gilts were removed from sows at d 5 of age and fed a milk-based liquid formula in until 14–16 d of age before being sacrificed for tissue collection. Three epithelial cell fractions, representing cells from the upper villus, the middle villus and the crypt regions, were sequentially isolated, with cell viability of 92–95% as assessed by trypan blue exclusion, along the crypt-villus axis from the entire jejunum by the distended sac method. Real time RT-PCR analyses (SmartCycler) with SYBR Green-I detection kit were used to examine SGLT1 mRNA abundance using β-actin as the house-keeping control. No differences (P>0.05) were observed in SGLT1 mRNA abundance between the upper and the middle villus cells but a lower (P<0.05) SGLT1 mRNA level was observed in the crypt cells. These results suggest that the SGLT1 gene expression is low in crypt cells and increases with cell differentiation in the villus cells in the formula-fed neonatal pigs.

A 96-well automated method to study inhibitors of human sodium-dependent D-glucose transport

The sodium-dependent D-glucose transporter (SGLT) family is involved in glucose uptake via intestinal absorption (SGLT1) or renal reabsorption (SGLT1 and SGLT2). Current methods for the screening of inhibitors of SGLT transporters are complex, expensive and very labor intensive, and have not been applied to human SGLT transporters. The purpose of the present study was to develop an alternative 96-well automated method to study the activity of human SGLT1 and SGLT2. Chinese hamster ovary (CHO) Flp-In cells were stably transfected with pcDNA5-SGLT1 or pcDNA5-SGLT2 plasmid and maintained in hygromycin-selection Ham's F12 culture medium until hygromycin-resistant clones were developed. SGLT1 and SGLT2 gene expression was evaluated by relative real-time reverse transcription-polymerase chain reaction (RT-PCR) quantification, Western blotting, and immunocytochemical analysis. The clones with higher expression of SGLT1 and SGLT2 were used for transport studies using [14C]-methyl-alpha-D-glucopyranoside ([14C]AMG). The advantage of using the 96-well format is the low amount of radioactive compounds and inhibitory substances required, and its ability to establish reproducibility because repetition into the assay. This method represents an initial approach in the development of transport-based high-throughput screening in the search for inhibitors of glucose transport. The proposed method can easily be performed to yield quantitative data regarding key aspects of glucose membrane transport and kinetic studies of potential inhibitors of human SGLT1 and SGLT2.

Glucose transporter gene expression in freshly isolated and cultured rat pneumocytes.

Alveolar epithelium in situ takes up luminal glucose by cotransport with sodium. Cultured alveolar type II pneumocytes have only sodium-independent glucose uptake. It is unclear which isoforms are responsible for glucose transport in these cells and why sodium-glucose cotransport activity disappears during culture. GLUT1, GLUT4, GLUT5 and SGLT1 mRNA were detected in freshly isolated rat alveolar type II cells by reverse transcriptase-polymerase chain reaction. We show that SGLT1 mRNA was 90% lower in cells cultured in plastic wells for 2 or 4 days than in freshly isolated cells. mRNAs coding for the facilitated transporters were reduced from 40% (GLUT1) and 75% (GLUT4 and GLUT5) in cultured cells. Cells cultured at the air-liquid interface better preserved their phenotype as attested by significantly higher surfactant-associated protein mRNA levels. However, these cells had no higher GLUT1 and SGLT1 gene expression. Thus, alveolar type II cells lose sodium-glucose cotransport activity in part because of a decrease in mRNA levels. These changes in gene expression and/or mRNA stability may be an additional consequence of the shift towards the type I cell phenotype observed in cultured type II pneumocytes.

Chronic acarbose-feeding increases GLUT1 protein without changing intestinal glucose absorption function

As α-glucosidase inhibitor, the antidiabetic drug acarbose reduces postprandial glucose levels by retarding the intestinal digestion of polysaccharides. However, it is unknown if acarbose also affects the expression of intestinal glucose transporters, especially the Na +-glucose cotransporter (SGLT1) and the glucose transporters GLUT1 and GLUT2. To unravel this question, Wistar rats received standard powdered chow either without (control) or with acarbose (40 mg acarbose/100 g chow) for 40 days. While food intake was slightly enhanced by acarbose, the drug had no influence on weight gain or plasma glucose and insulin levels. The acarbose-treatment did not alter the SGLT1 and GLUT2 gene expression in both upper and middle small intestine, whereas GLUT1 protein was increased by 75% in middle small intestine. Despite the territorial change in GLUT1 protein, the intestinal glucose absorption in an acarbose-free perfusion study was unaltered. In conclusion, the chronic use of acarbose did not alter the acarbose-free glucose absorption profile.

Hepatocyte growth factor up-regulates SGLT1 and GLUT5 gene expression after massive small bowel resection

Background/Purpose: Hepatocyte growth factor (HGF), originally known to stimulate hepatocyte DNA synthesis, also has been shown to stimulate growth of intestinal epithelial cells in vitro. The authors recently demonstrated that HGF can dramatically increase substrate absorption beyond the normal adaptive response after massive small bowel resection in the rat. However, the mechanism for this enhanced substrate absorption is unknown. This study was designed to determine if up-regulation of gene expression by HGF of the Na +/glucose cotransporter SGLT1 and the facilitative glucose transporter GLUT5 is a possible mechanism of action. Methods: Young adult Sprague-Dawley rats underwent an 80% small bowel resection and jejunostomy tube placement. Seven days later, an osmotic minipump was connected to the subcutaneously placed jejunostomy tube. The rats were divided into two groups based on the contents in the minipumps: group 1, saline (control, n = 5); and group 2, HGF at 75 μg/kg/d (n = 5). After a 14-day infusion, biopsy specimens of the small bowel mucosa were obtained. After total RNA extraction, Northern blot analysis was performed with SGLT1 and GLUT5 cDNA probes. Auto radiographs were quantitated by image analysis. Results: SGLT1 mRNA levels were significantly up-regulated in the HGF-treated animals (121% increase, P < .01) when compared with the control. Up-regulation of GLUT5 mRNA levels was also seen in the HGF-treated animals (96% increase, P < .01). Conclusions: These data, demonstrating that HGF up-regulates intestine epithelial glucose transporter gene expression after massive small bowel resection, may elucidate a mechanism of action for the enhanced carbohydrate absorption after HGF administration. This growth factor may be useful for patients with short bowel syndrome.