SGLT2 Activity Research Articles

Effect of Jardiance on glucose uptake into astrocytomas

PurposeSGLT2, the sodium glucose cotransporter two, is expressed in human pancreatic, prostate and brain tumors, and in a mouse cancer model SGLT2 inhibitors reduce tumor glucose uptake and growth. In this study we have measured the effect of a specific SGLT2 inhibitor, Jardiance® (Empagliflozin), on glucose uptake into astrocytomas in patients.MethodsWe have used a specific SGLT glucose tracer, α-methyl-4-[18F]fluoro-4-deoxy-α-D-glucopyranoside (Me4FDG), and Positron Emission Tomography (PET) to measure glucose uptake. Four of five patients enrolled had WHO grade IV glioblastomas, and one had a low grade WHO Grade II astrocytoma. Two dynamic brain PET scans were conducted on each patient, one before and one after treatment with a single oral dose of Jardiance, a specific SGLT2 inhibitor. As a control, we also determined the effect of oral Jardiance on renal SGLT2 activity.ResultsIn all five patients an oral dose (25 or 100 mg) of Jardiance reduced Me4FDG tumor accumulation, highly significant inhibition in four, and inhibited SGLT2 activity in the kidney.ConclusionsThese initial experiments show that SGLT2 is a functional glucose transporter in astocytomas, and Jardiance inhibited glucose uptake, a drug approved by the FDA to treat type 2 diabetes mellitus (T2DM), heart failure, and renal failure. We suggest that clinical trials be initiated to determine whether Jardiance reduces astrocytoma growth in patients.

149-OR: Genetic Modulation of HNF1A Activity via SGLT2 Deficiency Leads to Transient Intermittent Hyperglycemia: Consequences for HNF1A-MODY

Hepatocyte Nuclear Factor-1A (HNF1A) is a master regulator of key glucose-responsive genes in metabolic organs such as the intestine, liver, kidney, and pancreas. Heterozygous mutations in HNF1A cause Maturity-Onset-Diabetes-of-the-Young (HNF1A-MODY) . Mutant carriers are normoglycemic in childhood, but with age, they develop hyperglycemia. Although insulin secretory defects have been extensively studied, it is unclear whether HNF1A deficiency contributes to a progressive decline in alpha cell function. HNF1A-MODY patients display glycosuria and endogenous glucose production linked to reduced renal SGLT2 expression. Since SGLT2 is also expressed in alpha cells and is required for glucagon regulation, we hypothesized that HNF1A coordinates renal and alpha cell SGLT2 activity to maintain glucose homeostasis. Here, we studied Hnf1a regulation of renal Sglt2, Sglt1, and Glut2 in Hnf1a-/- mutant mice, glucagon expression and secretion in siHNF1A transfected human islets and we developed a novel Hnf1a mutant mouse model (Hnf1a+/Δe4-10) . Sglt2 protein was significantly reduced in the kidney of Hnf1a-/- mice, with no changes in Sglt1 or Glut2 protein expression. We observed that HNF1A is heterogeneously expressed in alpha, beta, and delta cells. siHNF1A islets displayed reduced insulin secretion and content, which was rescued by GLP1 treatment. HNF1A deficiency reduced SGLT2 levels, correlating with increased glucagon content and secretion. The Hnf1a+/Δe4-mice were born normoglycemic but became glucose intolerant at 12 weeks of age, mimicking the clinical characteristics of HNF1A-MODY patients. Collectively, these findings indicate that HNF1A deficiency downregulates renal and alpha cell SGLT2 expression, which is associated with an alteration in glucagon secretion in response to glucose stimulation. The new Hnf1a+/Δe4-mouse model may be a useful tool to study the progression of MODY with and without chronic drug treatment. Disclosure A.Acosta-montalvo: None. V.Gmyr: None. M.Cnop: None. J.A.Kerr-conte: None. F.Pattou: None. M.Pontoglio: None. A.Liston: None. C.Bonner: None. C.Saponaro: None. J.Thevenet: None. M.Chiral: None. A.Piron: None. N.Delalleau: None. G.Pasquetti: None. A.Coddeville: None. M.Moreno: None. Funding EFSD/Lilly (2017) awarded to Caroline Bonner, I-SITE ULNE awarded to Caroline Bonner, the European Genomic Institute for Diabetes (ANR-10-LABX-46) , the European Consortium for Islet Transplantation funded by the Juvenile Diabetes Research Foundation. TIGER was developed within the European Union’s Horizon 2020 research and innovation program project T2DSystems, under grant agreement 667191 (to Miriam Cnop) .

Central administration of sodium-glucose cotransporter-2 inhibitors increases food intake involving adenosine monophosphate-activated protein kinase phosphorylation in the lateral hypothalamus in healthy rats

IntroductionSodium glucose cotransporter-2 (SGLT2) inhibitors are widely used for diabetes treatment. Although SGLT2 inhibitors have been clinically observed to increase food intake, roles or even the presence of SGLT2 in...

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.

Effect of Insulin on Proximal Tubules Handling of Glucose: A Systematic Review.

Renal proximal tubules reabsorb glucose from the glomerular filtrate and release it back into the circulation. Modulation of glomerular filtration and renal glucose disposal are some of the insulin actions, but little is known about a possible insulin effect on tubular glucose reabsorption. This review is aimed at synthesizing the current knowledge about insulin action on glucose handling by proximal tubules. Method. A systematic article selection from Medline (PubMed) and Embase between 2008 and 2019. 180 selected articles were clustered into topics (renal insulin handling, proximal tubule glucose transport, renal gluconeogenesis, and renal insulin resistance). Summary of Results. Insulin upregulates its renal uptake and degradation, and there is probably a renal site-specific insulin action and resistance; studies in diabetic animal models suggest that insulin increases renal SGLT2 protein content; in vivo human studies on glucose transport are few, and results of glucose transporter protein and mRNA contents are conflicting in human kidney biopsies; maximum renal glucose reabsorptive capacity is higher in diabetic patients than in healthy subjects; glucose stimulates SGLT1, SGLT2, and GLUT2 in renal cell cultures while insulin raises SGLT2 protein availability and activity and seems to directly inhibit the SGLT1 activity despite it activating this transporter indirectly. Besides, insulin regulates SGLT2 inhibitor bioavailability, inhibits renal gluconeogenesis, and interferes with Na+K+ATPase activity impacting on glucose transport. Conclusion. Available data points to an important insulin participation in renal glucose handling, including tubular glucose transport, but human studies with reproducible and comparable method are still needed.

P1.11-14 SGLT2 Is a Diagnostic and Therapeutic Target for Early-Stage Lung Adenocarcinoma

Early diagnosis of lung adenocarcinoma (LUAD) is crucial. The National Lung Screening Trial showed a 20% reduction in lung cancer mortality in high risk individuals using low-dose helical computed tomography (CT). CT is highly sensitive for detecting lung nodules, but is limited by low specificity, especially for LUAD. On CT, LUAD may appear as solid or subsolid nodules. Most subsolid nodules are not cancer, and many will remain stable or resolve; however, subsolid lesions can represent premalignancy or adenocarcinoma in situ. These lesions in the early spectrum of LUAD may persist for months to years before transforming into invasive disease. As a result, current standard of care is to follow these patients with CT imaging to monitor these indeterminate lesions for radiologic signs of malignant progression. The identification of novel biomarkers to predict the malignant potential of these nodules at their initial identification is of paramount importance. We have recently discovered that premalignant ad early invasive lesions of the LUAD spectrum rely on sodium-glucose transporter 2 (SGLT2) for glucose uptake, whereas advanced carcinomas up-regulate transporters of the GLUT family. This is consistent with the observation that positron emission tomography (PET) with 2-[18F] fluorodeoxyglucose (FDG), which detects GLUT but not SGLT activity, is a standard tool for staging advanced disease, but has low sensitivity for early-stage LUAD. We measured SGLT2 activity in vivo with the PET tracer methyl-4-[18F] fluorodeoxyglucose (Me4FDG). Me4FDG detects early-stage, FDG-negative LUAD in mouse models and in patients. Importantly, Me4FDG uptake correlates with tumor growth rate in patient-derived LUAD xenografts (fig. 1). Targeting SGLT2 with FDA-approved inhibitors significantly reduces tumor growth and prolongs survival in genetic and patient-derived murine models, confirming an important role of SGLT2 in early-stage LUAD. SGLT2 is a promising biomarker not only to diagnose early-stage tumors by PET imaging, but also to predict response to SGLT2 inhibitors.

Role of sodium/glucose cotransporter inhibition on a rat model of angiotensin II\u2013dependent kidney damage

BackgroundRenal proximal tubular sodium and glucose reabsorption are regulated by the sodium-glucose cotransporter (SGLT2). Changes in this transporter can play a role in hyperglycaemia and reactive oxygen species (ROS) production. We demonstrated increased glucose absorption in proximal tubule membrane vesicles and increased expression of SGLT2 in hypertensive rats. Here we investigated Angiotensin II (Ang II) -dependent SGLT2 expression induction and the role of SGLT2 induction in the development of Ang II-dependent kidney damage. The aim of this study was to determine whether SGLT2 induction by Ang II is associated with Ang II-dependent kidney damage. We propose the following objectives a) to demonstrate that Ang II induces SGLT2 expression and b) to demonstrate that prevention of SGLT2 expression and activity prevent Ang II-induced kidney damage.MethodsWe used chronic Ang II infusion as a model of kidney damage in male Wistar rats and evaluated systolic blood pressure by telemetric methods. SGLT2 mRNA and protein expression were evaluated by PCR and immunoblotting. SGLT2 activity was evaluated in brush border membrane vesicles by measuring glucose uptake. ROS production was measured by confocal microscopy. The glomerular filtration rate (GFR) was evaluated by the inulin excretion method, and urinary protein excretion was evaluated by the Bradford method. Biological parameter evaluations were performed, after two weeks of infusion of Ang II. We compared the effects of Angiotensin II (AT1) receptor blockade by Losartan and SGLT2 inhibition by Empagliflozin both as monotherapy treatments and in combination on the development of kidney damage.ResultsChronic Ang II infusion led to a blood pressure elevation and increased SGLT2 mRNA expression and activity as well as kidney damage, as reflected by increased ROS production, decreased GFR and increased urinary protein excretion. AT1 receptor blockade prevented all these changes. By contrast, SGLT2 inhibition did not affect blood pressure and had a small effect on kidney damage. However, the combination of both drugs resulted in the potentiation of the effects observed by AT1 receptor blockade alone.ConclusionsWe suggest that Ang II-dependent increased SGLT2 induction is one mechanism by which Ang II induces kidney damage.

Abstract 766: SGLT-Mediated Na + Overload Results in Oxidative Stress and Abnormal SR Ca 2+ Release in Diabetic Hearts

Intracellular Na + concentration ([Na + ] i ) regulates Ca 2+ cycling, oxidative state and electrical stability of the heart. [Na + ] i is linked to glucose uptake through the Na + -glucose cotransporter (SGLT). The SGLT1 isoform is present in the heart and its overexpression causes hypertrophy and left-ventricular dysfunction. Here, we hypothesized that cardiac SGLT activity is increased in type-2 diabetes (T2D), which causes myocyte Na + overload and results in oxidative stress and exacerbated sarcoplasmic reticulum (SR) Ca 2+ leak. To test this hypothesis, we analyzed myocardial tissue from humans with/without T2D and compared rats with late-onset T2D that display diabetic cardiomyopathy similar to that seen in humans (HIP rats) with their non-diabetic littermates (WT). SGLT1 expression was increased in hearts from T2D patients compared to non-diabetic individuals and in hearts from HIP vs . WT rats. SGLT inhibition significantly decreased the uptake of both glucose and Na + in myocytes from HIP rats but not in WT, which indicates that SGLT function is increased in T2D hearts. While SGLT1 upregulation may partially compensate for the reduced insulin-dependent glucose uptake, the ensuing raise in Na + influx resulted in elevated [Na + ] i in HIP myocytes (by 3 mM compared to WT). Higher [Na + ] i causes oxidative stress by activating the mitochondrial Na + /Ca 2+ exchanger (mitoNCX), which lowers mitochondrial [Ca 2+ ] and thus slows down regeneration of the antioxidant NADPH. In agreement with a role for elevated [Na + ] i in causing oxidative stress in T2D hearts, H 2 O 2 production was increased in HIP myocytes vs . WT and mitoNCX inhibition, which uncouples mitochondria from [Na + ] i , significantly reduced H 2 O 2 production in HIP hearts. Oxidative stress enhances the SR Ca 2+ leak directly through oxidation of ryanodine receptors (RyRs) and indirectly via CaMKII activation and consequent RyR phosphorylation. SR Ca 2+ leak was augmented in myocytes from HIP vs . WT rats and mitoNCX inhibition significantly reduced the leak in HIP myocytes, but not in the WT. Thus, our data indicate that SGLT activity is enhanced in myocytes from T2D rats, which increases Na + influx and causes Na + overload. Elevated [Na + ] i contributes to oxidative stress and abnormal SR Ca 2+ leak in diabetic hearts.

Elevated glucose concentration in culture media decreases membrane trafficking of SGLT2 in LLC-PK1 cells via a cAMP/PKA-dependent pathway.

Na+-dependent glucose reabsorption in the renal proximal tubule is dynamically regulated by changes in blood glucose levels. There is, however, a disparity in reports studying the relationship between hyperglycemia and Na+-glucose-linked transporter (SGLT) function and expression. Similarly, manipulation of the glucose content in growth media of cultured renal cells has been shown to influence SGLT activity. In this investigation, SGLT activity was significantly lower in proximal tubule LLC-PK1 cells cultured in medium containing 17.5 than 5 mM glucose. α-Methyl d-glucopyranoside (AMG) transport kinetics showed reduced apparent Vmax and Km in cells grown in 17.5 mM glucose. SGLT2 was identified as the isoform responsible for glucose transport, and protein expression analyses showed decreased apical membrane localization of SGLT2 in cells grown in 17.5 mM glucose, explaining the reduced activity. Multiple signaling pathways have been implicated in regulation of SGLT activity and trafficking. Elevated media glucose decreased intracellular cAMP and PKA activation, leading to decreased SGLT2 trafficking into the plasma membrane, which was reversed after treatment with 1 µM forskolin. The effects of media glucose on SGLT activity were found to be dependent on p38 MAPK activation due to PKA-mediated signaling. Glucose-modulated AMG uptake is reversible and was associated with altered SGLT2 membrane trafficking and cAMP alterations. In summary, elevated glucose concentrations in culture medium decrease SGLT activity in LLC-PK1 cells by reducing membrane trafficking of SGLT2 via decreasing intracellular cAMP, resulting in a lowered PKA-dependent phosphorylation of p38 MAPK.

No direct effect of SGLT2 activity on glucagon secretion.

Sodium-glucose cotransporter (SGLT) 2 inhibitors constitute a new class of glucose-lowering drugs, but they increase glucagon secretion, which may counteract their glucose-lowering effect. Previous studies using static incubation of isolated human islets or the glucagon-secreting cell line α-TC1 suggested that this results from direct inhibition of alpha cell SGLT1/2-activity. The aim of this study was to test whether the effects of SGLT2 on glucagon secretion demonstrated in vitro could be reproduced in a more physiological setting. We explored the effect of SGLT2 activity on glucagon secretion using isolated perfused rat pancreas, a physiological model for glucagon secretion. Furthermore, we investigated Slc5a2 (the gene encoding SGLT2) expression in rat islets as well as in mouse and human islets and in mouse and human alpha, beta and delta cells to test for potential inter-species variations. SGLT2 protein content was also investigated in mouse, rat and human islets. Glucagon output decreased three- to fivefold within minutes of shifting from low (3.5mmol/l) to high (10mmol/l) glucose (4.0 ± 0.5pmol/15min vs 1.3 ± 0.3pmol/15min, p < 0.05). The output was unaffected by inhibition of SGLT1/2 with dapagliflozin or phloridzin or by addition of the SGLT1/2 substrate α-methylglucopyranoside, whether at low or high glucose concentrations (p = 0.29-0.99). Insulin and somatostatin secretion (potential paracrine regulators) was also unaffected. Slc5a2 expression and SGLT2 protein were marginal or below detection limit in rat, mouse and human islets and in mouse and human alpha, beta and delta cells. Our combined data show that increased plasma glucagon during SGLT2 inhibitor treatment is unlikely to result from direct inhibition of SGLT2 in alpha cells, but instead may occur downstream of their blood glucose-lowering effects.

2394 Sodium-glucose transporter 2 is a novel diagnostic and therapeutic target for early-stage lung adenocarcinoma

OBJECTIVES/SPECIFIC AIMS: Lung cancer claims 160,000 lives in the United States every year, and lung adenocarcinoma (LADC) is the most frequent type. Early diagnosis is crucial. Computed tomography (CT) is very sensitive in identifying early-stage lung nodules, but has low specificity. Increased glucose uptake is a hallmark of cancer measurable in vivo by fluorodeoxyglucose (FDG) positron-emission tomography (PET). FDG PET is widely used for cancer staging but has low sensitivity in the diagnosis of solitary lung nodules. We have previously identified an alternative glucose transporter, SGLT2, expressed in different types of cancer but not detected by FDG PET. SGLT2 activity can be measured in vivo with the PET tracer methyl-4-fluorodeoxyglucose (Me4FDG). The objective of this study was to test the hypothesis that SGLT2 is a novel diagnostic and therapeutic target in FDG-negative, early stage LADC. METHODS/STUDY POPULATION: To study glucose transporter expression in LADC, we performed immunohistochemistry with SGLT2- and GLUT1-specific antibodies in human lung pre-malignant lesions and LADC samples. To verify the possibility of detecting SGLT2 activity in vivo, we performed microPET imaging with the SGLT-specific tracer Me4FDG in a Kras-driven, p53-null genetically engineered mouse model and in patient-derived xenografts of LADC. Finally, we performed therapeutic trials in genetically engineered and patient-derived mouse models of LADC with the FDA-approved SGLT2 inhibitor canagliflozin. RESULTS/ANTICIPATED RESULTS: We observed a switch in the modality of glucose transport during lung carcinogenesis: SGLT2 was highly expressed in pre-malignant lesions and well-differentiated LADC, whereas GLUT1 was upregulated in advanced, poorly differentiated lesions. This pattern was observed both in human samples and in murine models. This observation led us to hypothesize that early-stage LADCs are often negative on FDG PET because this imaging modality does not detect the activity of SGLT2, which is expressed in early lesions. Therefore, we performed PET imaging with the tracer Me4FDG, that measures SGLT2 activity, in our mouse model, and observed that Me4FDG accumulated in small nodules that were negative with FDG. We confirmed the functionality of SGLT2 in human LADC by Me4FDG PET in patient-derived xenografts. To investigate the role of SGLT2-mediated glucose uptake in the early stages of LADC development, we treated both genetically engineered mice and patient-derived xenografts with FDA-approved SGLT2 inhibitors, showing that SGLT2 inhibition effectively reduced LADC growth and prolonged survival in mouse models. In addition, Me4FDG uptake predicted response to SGLT2 inhibition. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results show that sodium-dependent glucose transport is a critical metabolic supply strategy in the early stages of lung adenocarcinoma development, and that Me4FDG is a novel biomarker of early LADC and of SGLT-dependent tumor growth. The discovery of SGLT2 in LADC highlighted the need for a re-interpretation of FDG-negative lung nodules, which might rely on SGLT2 for glucose uptake, and therefore may be detected by the new tracer Me4FDG. We anticipate our findings will lead to clinical studies evaluating Me4FDG as a diagnostic tracer for solitary lung nodules and early LADC, and as a biomarker for the selection of patients eligible for treatment with SGLT2 inhibitors.

OS 17-03 ENHANCED SGLT2 FUNCTION REDUCES NATRIURESIS IN DIABETIC MICE

Objective: Hypertension is exacerbated in diabetic patients because of increased salt retention. The aim of the present study is to determine whether enhanced SGLT2 function accounts for salt retention in diabetic mice. Design and Method: 2-month type 2 diabetic db/db mice and their lean db/+ littermates were fed with normal (0.4% NaCl, ND) or high salt (8% NaCl, HS) diet for 6 months. The expression of SGLT2 was assayed by quantitative real-time PCR and western blot, and the acute SGLT2 activity assay was performed using treatment of a SGLT2 inhibitor, dapagliflozin (1mg/kg body weight), after glucose substitution (2 g/kg body weight) in the metabolic cages. The 24h-urinary sodium excretion and glycosuria were detected. Results: The body weight, fasting blood glucose and systolic blood pressure were higher in db/db mice than in db/+ mice. High salt intake not only increased systolic blood pressure and natriuresis, but also enhanced urinary volume and glycosuria in db/db mice (urinary volume: db/+ 186% vs. db/db 67%; urinary sodium excretion: db/+ 142% vs. db/db 57%). The renal SGLT2 mRNA and protein expression were noticeably higher in db/db mice than in db/+ mice, which can be down-regulated by high sodium intake. In response to dapagliflozin treatment, high sodium intake increased natriuresis and glycosuria to a much lesser extent in db/db mice than in db/+ mice (urinary sodium excretion increase: db/+ 170% vs. db/db 79%; urinary glucose excretion increase: db/+ 43% vs. db/db 15%). Conclusions: These results indicate that high sodium intake-induced natriuresis is hampered in diabetic mice and a reduction in natriuresis due to increased SGLT2 activity occurred in diabetes under high sodium intake.

MAP17 Is a Necessary Activator of Renal Na+/Glucose Cotransporter SGLT2.

The renal proximal tubule reabsorbs 90% of the filtered glucose load through the Na+-coupled glucose transporter SGLT2, and specific inhibitors of SGLT2 are now available to patients with diabetes to increase urinary glucose excretion. Using expression cloning, we identified an accessory protein, 17 kDa membrane-associated protein (MAP17), that increased SGLT2 activity in RNA-injected Xenopus oocytes by two orders of magnitude. Significant stimulation of SGLT2 activity also occurred in opossum kidney cells cotransfected with SGLT2 and MAP17. Notably, transfection with MAP17 did not change the quantity of SGLT2 protein at the cell surface in either cell type. To confirm the physiologic relevance of the MAP17-SGLT2 interaction, we studied a cohort of 60 individuals with familial renal glucosuria. One patient without any identifiable mutation in the SGLT2 coding gene (SLC5A2) displayed homozygosity for a splicing mutation (c.176+1G>A) in the MAP17 coding gene (PDZK1IP1). In the proximal tubule and in other tissues, MAP17 is known to interact with PDZK1, a scaffolding protein linked to other transporters, including Na+/H+ exchanger 3, and to signaling pathways, such as the A-kinase anchor protein 2/protein kinase A pathway. Thus, these results provide the basis for a more thorough characterization of SGLT2 which would include the possible effects of its inhibition on colocalized renal transporters.

SGLT2 inhibitors act from the extracellular surface of the cell membrane.

SGLT2 inhibitors are a new class of drugs that have been recently developed to treat type II diabetes. They lower glucose levels by inhibiting the renal Na+/glucose cotransporter SGLT2, thereby increasing the amount of glucose excreted in the urine. Pharmacodynamics studies have raised questions about how these inhibitors reach SGLT2 in the brush border membrane of the S1 and S2 segments of the renal proximal tubule: are these drugs filtered by the glomerulus and act extracellularly, or do they enter the cell and act intracellularly? To address this question we expressed hSGLT2 in HEK‐293T cells and determined the affinity of a specific hSGLT2 inhibitor, TA‐3404 (also known as JNJ‐30980924), from the extra‐ and intracellular side of the plasma membrane. Inhibition of SGLT2 activity (Na+/glucose currents) by TA‐3404 was determined using the whole‐cell patch clamp that allows controlling the composition of both the extracellular and intracellular solutions. We compared the results to those obtained using the nonselective SGLT inhibitor phlorizin, and to the effect of TA‐3404 on hSGLT1. Our results showed that TA‐3404 is a potent extracellular inhibitor of glucose inward SGLT2 transport (IC50 2 nmol/L) but it was ineffective from the intracellular compartment at both low (5 mmol/L) and high (150 mmol/L) intracellular NaCl concentrations. We conclude that TA‐3404 only inhibits SGLT2 from the extracellular side of the plasma membrane, suggesting that it is filtered from the blood through the glomerulus and acts from within the tubule lumen.

Functional Role of Glucose Metabolism, Osmotic Stress, and Sodium-Glucose Cotransporter Isoform-Mediated Transport on Na+/H+ Exchanger Isoform 3 Activity in the Renal Proximal Tubule

Na(+)-glucose cotransporter 1 (SGLT1)-mediated glucose uptake leads to activation of Na(+)-H(+) exchanger 3 (NHE3) in the intestine by a process that is not dependent on glucose metabolism. This coactivation may be important for postprandial nutrient uptake. However, it remains to be determined whether SGLT-mediated glucose uptake regulates NHE3-mediated NaHCO3 reabsorption in the renal proximal tubule. Considering that this nephron segment also expresses SGLT2 and that the kidneys and intestine show significant variations in daily glucose availability, the goal of this study was to determine the effect of SGLT-mediated glucose uptake on NHE3 activity in the renal proximal tubule. Stationary in vivo microperfusion experiments showed that luminal perfusion with 5 mM glucose stimulates NHE3-mediated bicarbonate reabsorption. This stimulatory effect was mediated by glycolytic metabolism but not through ATP production. Conversely, luminal perfusion with 40 mM glucose inhibited NHE3 because of cell swelling. Notably, pharmacologic inhibition of SGLT activity by Phlorizin produced a marked inhibition of NHE3, even in the absence of glucose. Furthermore, immunofluorescence experiments showed that NHE3 colocalizes with SGLT2 but not SGLT1 in the rat renal proximal tubule. Collectively, these findings show that glucose exerts a bimodal effect on NHE3. The physiologic metabolism of glucose stimulates NHE3 transport activity, whereas, supraphysiologic glucose concentrations inhibit this exchanger. Additionally, Phlorizin-sensitive SGLT transporters and NHE3 interact functionally in the proximal tubule.

SGLT2 : A Potential Target for the Pharmacogenetics of Type 2 Diabetes?

The kidney has attracted the attention of diabetologists as an organ involved in the regulation of glucose homeostasis not only by gluconeogenesis, but also by renal glucose excretion. Sodium-glucose cotransporters (SGLTs), particularly SGLT2, are responsible for reabsorption of up to 99% of the filtered glucose. SGLT2 is coded by the SLC5A2 gene, which maps on chromosome 16. Pharmacological reduction of tubular glucose reabsorption results in improved glycemic control in Type 2 diabetic patients. Since the SGLTs reabsorb most of the filtered glucose (90%), it is not surprising that mutations in SLC5A2 cause familial renal glucosuria. A recent study pointed out a possible role of common genetic variation in SLC5A2 in the control of glucose homeostasis. SLC5A2 polymorphisms might therefore represent potential candidates for pharmacogenomic studies targeting the impact of these variants on the efficacy of antidiabetic treatment that is based on inhibition of SGLT2 activity.

Regional distribution of SGLT activity in rat brain in vivo.

Na(+)-glucose cotransporter (SGLT) mRNAs have been detected in many organs of the body, but, apart from kidney and intestine, transporter expression, localization, and functional activity, as well as physiological significance, remain elusive. Using a SGLT-specific molecular imaging probe, α-methyl-4-deoxy-4-[(18)F]fluoro-D-glucopyranoside (Me-4-FDG) with ex vivo autoradiography and immunohistochemistry, we mapped in vivo the regional distribution of functional SGLTs in rat brain. Since Me-4-FDG is not a substrate for GLUT1 at the blood-brain barrier (BBB), in vivo delivery of the probe into the brain was achieved after opening of the BBB by an established procedure, osmotic shock. Ex vivo autoradiography showed that Me-4-FDG accumulated in regions of the cerebellum, hippocampus, frontal cortex, caudate nucleus, putamen, amygdala, parietal cortex, and paraventricular nucleus of the hypothalamus. Little or no Me-4-FDG accumulated in the brain stem. The regional accumulation of Me-4-FDG overlapped the distribution of SGLT1 protein detected by immunohistochemistry. In summary, after the BBB is opened, the specific substrate for SGLTs, Me-4-FDG, enters the brain and accumulates in selected regions shown to express SGLT1 protein. This localization and the sensitivity of these neurons to anoxia prompt the speculation that SGLTs may play an essential role in glucose utilization under stress such as ischemia. The expression of SGLTs in the brain raises questions about the potential effects of SGLT inhibitors under development for the treatment of diabetes.

Regulation of the human Na+-dependent glucose cotransporter hSGLT2

The human Na(+)-glucose cotransporter SGLT2 is expressed mainly in the kidney proximal convoluted tubule where it is considered to be responsible for the bulk of glucose reabsorption. Phosphorylation profiling has revealed that SGLT2 exists in a phosphorylated state in the rat renal proximal tubule cortex, so we decided to investigate the regulation of human SGLT2 (hSGLT2) by protein kinases. hSGLT2 was expressed in human embryonic kidney (HEK) 293T cells, and the activity of the protein was measured using radiotracer and whole cell patch-clamp electrophysiology assays before and after activation of protein kinases. 8-Bromo-adenosine cAMP (8-Br-cAMP) was used to activate protein kinase A, and sn-1,2-dioctanoylglycerol (DOG) was used to activate protein kinase C (PKC). 8-Br-cAMP stimulated D-[α-methyl-(14)C]glucopyranoside ([(14)C]α-MDG) uptake and Na(+)-glucose currents by 200% and DOG increased [(14)C]α-MDG uptake and Na(+)-glucose currents by 50%. In both cases the increase in SGLT2 activity was marked by an increase in the maximum rate of transport with no change in glucose affinity. These effects were completely negated by mutation of serine 624 to alanine. Insulin induced a 250% increase in Na(+)-glucose transport by wild-type but not S624A SGLT2. Parallel studies confirmed that the activity of hSGLT1 was regulated by PKA and PKC due to changes in the number of transporters in the cell membrane. hSGLT1 was relatively insensitive to insulin. We conclude that hSGLT1 and hSGLT2 are regulated by different mechanisms and suggest that insulin is an SGLT2 agonist in vivo.

Characterization of glucose and SGLT mediated effect on NHE3 in renal proximal tubule

We previously reported that proximal tubule perfusion with glucose 5mM stimulates Na+/H+ exchanger isoform 3 (NHE3) activity via p38MAPKinase pathway. Perfusions with higher glucose concentrations (up to 40 mM) were seen to inhibit NHE3 in comparison to a nominally glucose free solution. However, the role of SGLT1 and/or SGLT2 or glucose metabolism in these distinct effects on NHE3 has not been determined so far. We therefore performed microperfusion experiments with sugar free (CTRL), glucose 5mM (G5) and glucose 40mM (G40) plus 2mM Phloridzin (Plz, a SGLT inhibitor). Plz inhibited CTRL (46,2%) and G5 (60,7%) NHE3 activity but had no additional inhibitory effect on G40 perfusion, showing that SGLT activity is necessary for NHE3 function. In addition, the substitution of glucose by galactose (transported only by SGLT1) completely inhibited the G5 and G40 effect, suggesting that only SGLT2 activity or glucose metabolism modulates NHE3 activity. The substitution of glucose by Methyl α‐D‐glucopyranoside (non‐metabolizable glucose) abolished the G5 but not G40 effect, showing that G5 stimulates NHE3 through glucose metabolism, while G40 did not. We concluded that SGLT activity is necessary for NHE3 function, SGLT2 mediates the effects of both low and high glucose on NHE3 activity and the glucose metabolism is involved with the stimulatory effect of G5 on the transporter. Supported by FAPESP and CNPq

Antidiabetic Effects of SGLT2-Selective Inhibitor Ipragliflozin in Streptozotocin–Nicotinamide-Induced Mildly Diabetic Mice

Sodium-glucose cotransporter (SGLT) 2 plays an important role in renal glucose reabsorption, and inhibition of renal SGLT2 activity represents an innovative strategy for the treatment of hyperglycemia in diabetic patients. The present study investigated the antidiabetic effects of ipragliflozin, a SGLT2-selective inhibitor, in streptozotocin–nicotinamide-induced mildly diabetic mice, which exhibited a mild decline in glucose tolerance associated with the loss of early-phase insulin secretion. Oral administration of ipragliflozin increased urinary glucose excretion in a dose-dependent manner, an effect which was significant at doses of 0.3 mg/kg or higher and lasted over 12 h. In addition, ipragliflozin dose-dependently improved hyperglycemia and glucose intolerance with concomitant decreases in plasma insulin levels without causing hypoglycemia. Once-daily dosing of ipragliflozin (0.1 – 3 mg/kg) for 4 weeks attenuated hyperglycemia, glucose intolerance, and impaired insulin secretion. These results suggest that the SGLT2-selective inhibitor ipragliflozin increases urinary glucose excretion by inhibiting renal glucose reabsorption, improves hyperglycemia in streptozotocin–nicotinamide-induced mildly diabetic mice, and may be useful for treating type 2 diabetes.