Sodium-glucose Cotransporter 2 Expression Research Articles

Effects of SGLT2 Inhibition in Human Kidney Proximal Tubular Cells—Renoprotection in Diabetic Nephropathy?

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.

Pharmacodynamics and Subchronic Toxicity in Mice and Monkeys of ISIS 388626, a Second-Generation Antisense Oligonucleotide That Targets Human Sodium Glucose Cotransporter 2

ISIS 388626, a 2'-methoxyethyl (MOE)-modified antisense oligonucleotide (ASO) that targets human sodium glucose cotransporter 2 (SGLT2) mRNA, is in clinical trials for the management of diabetes. SGLT2 plays a pivotal role in renal glucose reabsorption, and inhibition of SGLT2 is anticipated to reduce hyperglycemia in diabetic subjects by increasing urinary glucose elimination. To selectively inhibit SGLT2 in the kidney, ISIS 388626 was designed as a "shortmer" ASO, consisting of only 12 nucleotides with two 2'-MOE-modified nucleotides at the termini. Mice and monkeys received up to 30 mg/kg/week ISIS 388626 via subcutaneous injection for 6 or 13 weeks. Dose-dependent decreases in renal SGLT2 mRNA expression were observed, which correlated with dose-related increases in glucosuria without concomitant hypoglycemia. There were no histologic changes in the kidney attributed to SGLT2 inhibition after 6 or 13 weeks of treatment. The remaining changes observed in these studies were typical of those produced in these species by the administration of oligonucleotides, correlated with high doses of ISIS 388626, and were unrelated to the inhibition of SGLT2 expression. The kidney contained the highest concentration of ISIS 388626, and dose-dependent basophilic granule accumulation in tubular epithelial cells of the kidney, which is evidence of oligonucleotide accumulation in these cells, was the only histologic change identified. No changes in kidney function were observed. These results revealed only readily reversible changes after the administration of ISIS 388626 and support the continued investigation of the safety and efficacy of ISIS 388626 in human trials.

Abstract 102: Cardiac Sodium/Glucose Cotransporter 1, a Novel Cardiac Glucose Transporter, Is Cardioprotective in Ischemia-Reperfusion Injury

Background: The expression of cardiac sodium/glucose cotransporter 1 (SGLT1) is up-regulated in ischemic heart disease. However, the regulation and the biological role of SGLT1 activity in cardiomyocytes are unclear. Hypothesis: We hypothesize that SGLT1 inhibition during ischemia-reperfusion (IR) will lead to worsening cardiac function and recovery. In addition we hypothesize that SGLT1 expression is upregulated by increased AMPK activity in ischemia. Methods: Transgenic mice with cardiac-specific RNAi knockdown of SGLT1 (TG SGLT1-KD ) were subjected to ex vivo IR (n > 3/group) using a Langendorff hanging heart preparation with 15 min of no-flow ischemia and 20 min of reperfusion while undergoing hemodynamic monitoring. FVB strain wild-type 8-12 week-old male mice (n > 3/group) were administered either the SGLT1 inhibitor phlorizin (400 mg/kg IP) or vehicle and subjected to in vivo ischemia for 30 min by ligation of the left anterior descending (LAD) coronary artery. Hearts were harvested 48 hours later. In vitro studies were conducted in HL-1 cardiomyocytes subjected to 1 h of simulated ischemia with 1 mM NaCN. HL-1 cells were also treated with 1 mM AICAR, an AMPK activator, for 16 hours and SGLT1 expression was measured. Results: In ex vivo IR studies, there was an impairment in the early recovery of double product in the TG SGLT1-KD mice compared to control mice (37% vs 92% at 1 min into reperfusion, P < 0.05). In in vivo IR studies, phlorizin led to a marked increase in oxidative stress documented by cardiac malondialdehyde (MDA) (control 1.00±0.00; IR 1.55±0.35; IR + phlorizin 16.15±0.40, P <0.001),an increase in apoptosis documented by TUNEL staining, and hemodynamic impairment: +dP/dt (mmHg/sec), control 11445±504, IR 9811±2613, IR + phlorizin 5139±481, P <0.05; -dP/dt (mmHg/sec), control -10625±589, IR -6770±1142, IR + phlorizin -4153±474, P <0.05. In HL-1 cardiomyocytes, there was a 6.7-fold increase in SGLT1 expression after simulated ischemia, and a 2.0-fold increase after AICAR exposure. Conclusion: SGLT1 expression increases when cardiomyocytes are subjected to ischemia, an effect that may be mediated by AMPK. Inhibition of SGLT1 leads to impaired myocardial recovery from ischemia.

Influence of age and Enterococcus faecium NCIMB 10415 on development of small intestinal digestive physiology in piglets

This study aimed at studying the effect of age and Enterococcus faecium NCIMB 10415 on jejunal physiological measures in suckling and weaned piglets. A total of 112 piglets from 16 landrace sows were used. Sows received either no probiotic (n=8, control) or were fed a diet with 4.2 to 4.3×106cfu/g E. faecium NCIMB 10415 (n=8, EF) from 28days ante partum onwards. Piglets were offered creep feed beginning at 12 d of age and piglets were weaned to a pre-starter diet at 26 d of age. For EF piglets, the creep feed (pre-starter) and starter diets contained 5.1×106cfu/g and 3.6×106cfu EF/g, respectively. Twenty-seven randomly selected piglets from each group were euthanized at 12 (n=6), 26 (n=6), 34 (n=7), and 54d (n=8) of age and a 45cm mid-jejunal segment was taken. Histomorphometry was performed and relative transcript abundance of caspase-3, proliferating cell nuclear antigen, intestinal alkaline phosphatase (IAP), lactase-phlorizin hydrolase (LPH), sucrase (SUC), aminopeptidase-N (APN) and sodium/glucose cotransporter 1 (SGLT1) was determined. Lactase (LAC), maltase (MAL), SUC, APN and IAP activities were measured in brush border membranes. The EF treatment did not affect (P>0.05) performance variables like the pre- or post-weaning daily weight gain, feed intake, incidence of diarrhoea and ileal and total tract nutrient digestibility, except the feed conversion ratio which was reduced (P=0.033). Jejunal villus length and crypt depth did not differ between groups (P>0.05) but increased age-dependently (P=0.003 and P<0.001, respectively). Feeding EF did not markedly influence brush border enzyme expression or activity. Enzyme activity increased (SUC, MAL) or decreased (LAC, IAP) with age reflecting maturation and adaptation to creep feed (P<0.001). The gene expression of SUC, SGLT1 and IAP changed over time (P=0.012, P=0.003 and P=0.013). Furthermore, gene expression was not affected by treatment. The current study suggests that EF did not markedly affect physiological variables associated with digestive function. Age of piglets seems to be the main factor determining function and morphology of the jejunal tissue, reflecting both maturation and adaptation to creep feed.

Sodium/Glucose Co-transporter 1 Expression Increases in Human Diseased Prostate

Sodium/glucose co-transporter 1 (SGLT1) is an active glucose transporter that takes up glucose into cells independent of the extracellular concentration of glucose. This transporter plays a critical role in maintaining glucose homeostasis at both physiological and pathological levels. The expression level of SGLT1 in normal and diseased human prostatic tissue has not been determined. We produced two rabbit polyclonal antibodies against human SGLT1, one each for immunohistochemical and Western blot analyses, and characterized the expression of SGLT1 in human prostate tissues: normal prostate (n=3), benign prostatic hyperplasia (BPH) (n=53), prostatic intraepithelial neoplasia (PIN) (n=9), and prostate cancer (PCa) (n=44). In normal prostate tissue, SGLT1 was weakly expressed exclusively in the epithelium. The transporter was significantly increased in the basal cells and stromal cells of BPH, increased in the epithelial cells of PIN, and frequently overexpressed in stromal cells and universally overexpressed in the tumor cells of PCa. The pattern of expression was shown as membranous/ cytoplasmic staining in low-grade cancer cells and nuclear envelope staining in high-grade cancer cells. The SGLT1-positive stromal cells of BPH and PCa tissues were negative for tenascin, a marker of reactive stromal cells. We concluded that SGLT1 is up-regulated in BPH and PCa, and SGLT1 may serve as a potential therapeutic target for treating these prostate disorders.

Abnormal expression and dysfunction of novel SGLT2 mutations identified in familial renal glucosuria patients

Familial renal glucosuria (FRG) is characterized by persistent glucosuria despite normal serum glucose and in the absence of overt tubular dysfunction. Mutation of sodium/glucose co-transporter 2 (SGLT2) has been identified and was recently reported to be involved in FRG. However, the functional and pathological consequences of such mutations remain unknown. In the current study, we collected four families with FRG. Sequencing of the SGLT2 coding region, intronic segments and cDNA revealed three missense mutations (294C>A: F98L; 1388T>G: L463R; 1435C>G: R479G) and two splice mutations (IVS 1-16 C>A: Del exon3; IVS 11+1 G>C: Del exon11). The probands were either heterozygous or compound heterozygous for SGLT2 mutations, and had glucosuria quantified at 6-27 g/day. Human 293 cells were transfected with the plasmid constructs to study the expression and function of SGLT2 mutants in vitro. Confocal microscopy using green fluorescent protein (GFP) revealed that the mutation results in a loss of punctate membrane pattern typical of the wild-type SGLT2 except in the 294C>A mutant. All mutants had significantly lower transport capacity in comparison to the wild-type control (26.49-71.48%). Renal biopsy in one consenting proband revealed significantly lower SGLT2 expression in the apical side of the proximal convoluted tubule in comparison to both healthy and disease controls (minimal change disease and diabetic nephropathy). The current study provides functional clues regarding the SGLT2 molecule from genotype to phenotype in FRG families.

SGLT1 protein expression in plasma membrane of acinar cells correlates with the sympathetic outflow to salivary glands in diabetic and hypertensive rats

Salivary gland dysfunction is a feature in diabetes and hypertension. We hypothesized that sodium-glucose cotransporter 1 (SGLT1) participates in salivary dysfunctions through a sympathetic- and protein kinase A (PKA)-mediated pathway. In Wistar-Kyoto (WKY), diabetic WKY (WKY-D), spontaneously hypertensive (SHR), and diabetic SHR (SHR-D) rats, PKA/SGLT1 proteins were analyzed in parotid and submandibular glands, and the sympathetic nerve activity (SNA) to the glands was monitored. Basal SNA was threefold higher in SHR (P < 0.001 vs. WKY), and diabetes decreased this activity (∼50%, P < 0.05) in both WKY and SHR. The catalytic subunit of PKA and the plasma membrane SGLT1 content in acinar cells were regulated in parallel to the SNA. Electrical stimulation of the sympathetic branch to salivary glands increased (∼30%, P < 0.05) PKA and SGLT1 expression. Immunohistochemical analysis confirmed the observed regulations of SGLT1, revealing its location in basolateral membrane of acinar cells. Taken together, our results show highly coordinated regulation of sympathetic activity upon PKA activity and plasma membrane SGLT1 content in salivary glands. Furthermore, the present findings show that diabetic- and/or hypertensive-induced changes in the sympathetic activity correlate with changes in SGLT1 expression in basolateral membrane of acinar cells, which can participate in the salivary glands dysfunctions reported by patients with these pathologies.

Sodium-Dependent Phosphate Uptake in the Jejunum Is Post-Transcriptionally Regulated in Pigs Fed a Low-Phosphorus Diet and Is Independent of Dietary Calcium Concentration

In rodents, severe dietary P restriction increases active phosphate absorption by the intestine. However, it remains unknown if moderate dietary P restriction has a similar effect. Weanling pigs (n = 32; body weight 7.4 ± 0.55 kg) were used in a 2 × 2 factorial design and fed dietary available P (aP) concentrations of 0.23 or 0.40% and Ca concentrations of 0.58 or 1.00% for 14 d. Diets were formulated on an aP basis instead of a total P basis, because pigs are unable to absorb phytate-P present in corn and soybean meal. Jejunal segments were mounted in modified Ussing chambers for determination of Na+-dependent nutrient transport. Intestinal mucosal scrapings were taken for RNA isolation and brush border membrane (BBM) vesicle isolation. Na+-dependent phosphate uptake and gene expression of Na-phosphate cotransporter IIb (NaPi-IIb), SGLT-1 (sodium/glucose cotransporter-1), and calbindin D(9k) and protein expression of NaPi-IIb were evaluated. Na+-dependent phosphate transport increased (P < 0.05) 46% as dietary aP concentration was decreased. However, increased Na+-dependent phosphate uptake was not accompanied by increased NaPi-IIb mRNA expression. Expression of NaPi-IIb protein in the BBM increased (P < 0.01) 84% in pigs fed low-P diets compared with pigs fed adequate-P diets. No dietary Ca effects or aP × Ca interactions were detected for Na-dependent P uptake, mRNA or protein expression of NaPi-IIb, or mRNA expression of calbindin D(9k). These data suggest that restricting dietary aP concentration by only 43% stimulates Na+-dependent phosphate uptake and expression of the NaPi-IIb protein in the BBM of the small intestine and through a post-transcriptional mechanism.

Diurnal expression of the rat intestinal sodium–glucose cotransporter 1 (SGLT1) is independent of local luminal factors

The intestinal sodium-glucose cotransporter 1 (SGLT1) is responsible for all secondary active transport of dietary glucose, and it presents a potential therapeutic target for obesity and diabetes. SGLT1 expression varies with a profound diurnal rhythm, matching expression to nutrient intake. The mechanisms entraining this rhythm remain unknown. We investigated the role of local nutrient signals in diurnal SGLT1 entrainment. Male Sprague-Dawley rats, which were acclimatized to a 12:12 light:dark cycle, underwent laparotomy with formation of isolated proximal jejunal loops (Thiry-Vella loops). Animals were recovered for 10 days before harvesting at 4 6-h intervals (Zeitgeber times ZT3, ZT9, ZT15, and ZT21, where ZT0 is lights on; n = 6-8). SGLT1 expression was assessed in protein, and mRNA extracts of mucosa were harvested from both isolated loops (LOOP) and remnant jejunum (JEJ). Isolated loops were healthy but atrophic with minimal changes to villus architecture. A normal anticipatory rhythm was observed in Sglt1 transcription in both LOOP and JEJ, with the peak signal at ZT9 (2.7-fold, P < .001). Normal diurnal rhythms were also observed in the protein signal, with peak expression in both LOOP and JEJ at ZT9 to 15 (2.1-fold, P < .05). However, an additional more mobile polypeptide band was also observed in all LOOP samples but not in JEJ samples (61 kDa vs 69 kDa). Enzymatic deglycosylation suggested this to be deglycosylated SGLT1. The persistence of SGLT1 rhythmicity in isolated loops indicates that diurnal induction is independent of local luminal nutrient delivery, and it suggests a reliance on systemic entrainment pathways. However, local luminal signals may regulate glycosylation and, therefore, the posttranslational handling of SGLT1.

Fecal Stream Is Essential for Adaptive Induction of Glucose-Coupled Sodium Transport in the Remnant Ileum After Total Proctocolectomy

Our previous studies demonstrated that sodium glucose cotransporter 1 (SGLT-1) was induced in the remnant ileum of total colectomized rats via the action of factors other than hyperaldosteronism. The aim of the present study was to clarify whether fecal stream is required for the enhancement of SGLT-1-mediated sodium transport. Twenty-seven pairs of ileal tissues were obtained from the proximal and distal side, respectively, of loop ileostomy after total proctocolectomy. Mucosae were mounted in an Ussing chamber to evaluate glucose-coupled sodium transport. Levels of SGLT-1 mRNA in proximal and distal mucosae were compared by Northern blotting. Villous height and crypt depth were measured to test for correlations between mucosal structure and SGLT-1-mediated sodium transport or mRNA expression levels. Both glucose-coupled sodium transport and expression of SGLT-1 mRNA were significantly lower in distal mucosae relative to proximal mucosae. In distal mucosae, villous height, but not crypt depth, was significantly lower than in proximal mucosae, demonstrating a positive correlation between villous height and SGLT-1 function and expression. Comparative studies of proximal and distal mucosae demonstrated that in addition to hormonal changes, fecal stream is required for full induction of the sodium transport system (which includes SGLT-1-mediated transport) in the remnant ileum following total proctocolectomy.

Effect of epidermal growth factor on small intestinal sodium/glucose cotransporter–1 expression in a rabbit model of intrauterine growth retardation

Introduction Intrauterine growth–retarded (IUGR) infants have impaired gastrointestinal function with feeding difficulties and predisposition to necrotizing enterocolitis. The rabbit provides a model of IUGR based on uterine position. Sodium/glucose cotransporter–1 (SGLT-1) is a membrane protein responsible for carbohydrate transport across the intestinal brush border membrane. Previous studies demonstrate increases in small intestinal (SI) nutrient uptake in response to amniotic fluid supplementation with epidermal growth factor (EGF). To determine whether SGLT-1 expression plays a role in the intestinal response to EGF supplementation, this IUGR rabbit model was evaluated. Methods Eight pregnant rabbits underwent placement of intraamniotic catheters into 2 normal (Nl) and 2 IUGR fetuses per mother on gestational day 24. Mini-osmotic pumps infused either EGF (300 μg/kg per day) or control solution forming 4 study groups (EGF-Nl vs Cont-Nl; EGF-IUGR vs Cont-IUGR). On gestational day 31, the fetal SI was harvested. Sodium/glucose cotransporter–1/glyceraldehyde-3-phosphate dehydrogenase messenger RNA (mRNA) densitometric band ratios were measured by reverse transcriptase polymerase chain reaction. Immunohistochemistry SGLT-1 staining was performed on middle SI. Statistical analysis was performed using the analysis of variance. Results Sodium/glucose cotransporter–1 was expressed in the gastrointestinal tract throughout the last one third of gestation. There were no native differences in SGLT-1 mRNA expression between Nl and IUGR fetuses at term. Epidermal growth factor infusion did not significantly affect SI SGLT-1 mRNA expression in either Nl or IUGR fetuses vs controls (EGF-Nl = 1.94 vs Cont-Nl = 1.94, P = .98; EGF-IUGR = 1.77 vs Cont-IUGR = 1.85, P = .74). Immunohistochemistry revealed increased SGLT-1 SI protein expression in infused IUGR fetuses. Conclusions Increases in previously documented up-regulation in SI nutrient transport after EGF infusion are independent of SGLT-1 mRNA expression. Further studies are warranted investigating SGLT-1 protein expression, localization, and functional kinetics in response to amniotic fluid supplementation with EGF.

Expression of glucose transporters in lactating human mammary gland epithelial cells.

Human milk contains 60-80 g/l lactose and and oligosaccharides. To synthesize this large amount of carbohydrates, the lactating mammary gland has a high demand for precursor molecules. such as glucose and galactose. In the present study we investigated the molecular basis for the uptake of glucose and galactose into the human mammary gland. Using RT-PCR, Southern and Western blotting we analyzed the expression of SGLT1 (sodium glucose cotransporter 1) und GLUT1 (sodium independent glucose transporter) in epithelial cells isolated from fresh human milk. Southern blot analysis of the amplicions revealed the expression of SGLT1 mRNA but not of GLUT1 mRNA in milk epithelial cells. Using Western blotting, SGLT1 protein was identified in human milk cells. CONCLUSIONS. Our findings indicate that 1) the cell fraction isolated from fresh human milk is a suitable model for investigating gene expression in the human mammary gland and 2) lactating human mammary gland epithelial cells are supplied with monosaccharides mainly via SGLT1.