Membrane potential accelerates sugar uptake by stabilizing the outward facing conformation of the Na/glucose symporter vSGLT.

Sodium-dependent glucose transporters (SGLTs) couple a downhill Na+ ion gradient to actively transport sugars. Here, we investigate the impact of the membrane potential on vSGLT structure and function using sugar uptake assays, double electron-electron resonance (DEER), electrostatic calculations, and kinetic modeling. Negative membrane potentials, as present in all cell types, shift the conformational equilibrium of vSGLT towards an outward-facing conformation, leading to increased sugar transport rates. Electrostatic calculations identify gating charge residues responsible for this conformational shift that when mutated reduce galactose transport and eliminate the response of vSGLT to potential. Based on these findings, we propose a comprehensive framework for sugar transport via vSGLT, where the cellular membrane potential facilitates resetting of the transporter after cargo release. This framework holds significance not only for SGLTs but also for other transporters and channels.

The Molecular Basis of Glucose Galactose Malabsorption in a Large Swedish Pedigree.

Glucose-galactose malabsorption (GGM) is due to mutations in the gene coding for the intestinal sodium glucose cotransporter SGLT1 (SLC5A1). Here we identify the rare variant Gln457Arg (Q457R) in a large pedigree of patients in the Västerbotten County in Northern Sweden with the clinical phenotype of GGM. The functional effect of the Q457R mutation was determined in protein expressed in Xenopus laevis oocytes using biophysical and biochemical methods. The mutant failed to transport the specific SGLT1 sugar analog α-methyl-D-glucopyranoside (αMDG). Q457R SGLT1 was synthesized in amounts comparable to the wild-type (WT) transporter. SGLT1 charge measurements and freeze-fracture electron microscopy demonstrated that the mutant protein was inserted into the plasma membrane. Electrophysiological experiments, both steady-state and presteady-state, demonstrated that the mutant bound sugar with an affinity lower than the WT transporter. Together with our previous studies on Q457C and Q457E mutants, we established that the positive charge on Q457R prevented the translocation of sugar from the outward-facing to inward-facing conformation. This is contrary to other GGM cases where missense mutations caused defects in trafficking SGLT1 to the plasma membrane. Thirteen GGM patients are now added to the pedigree traced back to the late 17th century. The frequency of the Q457R variant in Västerbotten County genomes, 0.0067, is higher than in the general Swedish population, 0.0015, and higher than the general European population, 0.000067. This explains the high number of GGM cases in this region of Sweden.

Function Trumps Form in Two Sugar Symporters, LacY and vSGLT.

Active transport of sugars into bacteria occurs through symporters driven by ion gradients. LacY is the most well-studied proton sugar symporter, whereas vSGLT is the most characterized sodium sugar symporter. These are members of the major facilitator (MFS) and the amino acid-Polyamine organocation (APS) transporter superfamilies. While there is no structural homology between these transporters, they operate by a similar mechanism. They are nano-machines driven by their respective ion electrochemical potential gradients across the membrane. LacY has 12 transmembrane helices (TMs) organized in two 6-TM bundles, each containing two 3-helix TM repeats. vSGLT has a core structure of 10 TM helices organized in two inverted repeats (TM 1-5 and TM 6-10). In each case, a single sugar is bound in a central cavity and sugar selectivity is determined by hydrogen- and hydrophobic- bonding with side chains in the binding site. In vSGLT, the sodium-binding site is formed through coordination with carbonyl- and hydroxyl-oxygens from neighboring side chains, whereas in LacY the proton (H3O+) site is thought to be a single glutamate residue (Glu325). The remaining challenge for both transporters is to determine how ion electrochemical potential gradients drive uphill sugar transport.

SGLT2 Inhibitors: Physiology and Pharmacology.

SGLTs are sodium glucose transporters found on the luminal membrane of the proximal tubule, where they reabsorb some 180 g (1 mol) of glucose from the glomerular filtrate each day. The natural glucoside phlorizin completely blocks glucose reabsorption. Oral SGLT2 inhibitors are rapidly absorbed into the blood stream, where theyremain in the circulation for hours. On glomerular filtration, they bind specifically to SGLT2 in the luminal membrane of the early proximal tubule to reduce glucose reabsorption by 50%-60%. Because of glucose excretion, these drugs lower plasma glucose and glycosylated hemoglobin levels in patients with type 2 diabetes mellitus. The drugs also protect against heart and renal failure. The aim of this review is to summarize what is known about the physiology of renal SGLTs and the pharmacology of SGLT drugs.

SGLT2 and cancer.

Glycolysis plays a central role in tumor metabolism and growth, and this is reflected in a high rate of glucose uptake. It is commonly assumed that the upregulation of the facilitated glucose transporter GLUT1 meets the tumor's demand for sugar. This underlies the success in using 2FDG PET imaging in the clinic to identify and stage many tumors. However, 2FDG is not a substrate for a second class of glucose transporters, the sodium-dependent glucose cotransporters, SGLTs, and so 2FDG PET may not provide a complete picture. A specific new radiotracer to detect SGLT activity has been introduced, Me4FDG, and this provides an opportunity to explore the potential role of SGLTs in supporting tumor glycolysis. In this brief review, I highlight the development of Me4FDG and our preliminary studies of Me4FDG PET in cancer patients. We find that the renal isoform, SGLT2, is expressed in pancreatic and prostate tumors and glioblastomas, and Me4FDG PET introduces a new method to image tumors. As SGLT2 drugs are successful in treating type 2 diabetes mellitus, they may also provide a new therapy.

Inhibitor binding mode and allosteric regulation of Na+-glucose symporters.

Sodium-dependent glucose transporters (SGLTs) exploit sodium gradients to transport sugars across the plasma membrane. Due to their role in renal sugar reabsorption, SGLTs are targets for the treatment of type 2 diabetes. Current therapeutics are phlorizin derivatives that contain a sugar moiety bound to an aromatic aglycon tail. Here, we develop structural models of human SGLT1/2 in complex with inhibitors by combining computational and functional studies. Inhibitors bind with the sugar moiety in the sugar pocket and the aglycon tail in the extracellular vestibule. The binding poses corroborate mutagenesis studies and suggest a partial closure of the outer gate upon binding. The models also reveal a putative Na+ binding site in hSGLT1 whose disruption reduces the transport stoichiometry to the value observed in hSGLT2 and increases inhibition by aglycon tails. Our work demonstrates that subtype selectivity arises from Na+-regulated outer gate closure and a variable region in extracellular loop EL5.

Physiology of renal glucose handling via SGLT1, SGLT2 and GLUT2.

The concentration of glucose in plasma is held within narrow limits (4-10 mmol/l), primarily to ensure fuel supply to the brain. Kidneys play a role in glucose homeostasis in the body by ensuring that glucose is not lost in the urine. Three membrane proteins are responsible for glucose reabsorption from the glomerular filtrate in the proximal tubule: sodium-glucose cotransporters SGLT1 and SGLT2, in the apical membrane, and GLUT2, a uniporter in the basolateral membrane. 'Knockout' of these transporters in mice and men results in the excretion of filtered glucose in the urine. In humans, intravenous injection of the plant glucoside phlorizin also results in excretion of the full filtered glucose load. This outcome and the finding that, in an animal model, phlorizin reversed the symptoms of diabetes, has stimulated the development and successful introduction of SGLT2 inhibitors, gliflozins, in the treatment of type 2 diabetes mellitus. Here we summarise the current state of our knowledge about the physiology of renal glucose handling and provide background to the development of SGLT2 inhibitors for type 2 diabetes treatment.

Intestinal absorption of glucose in mice as determined by positron emission tomography.

KEY POINTS: The goal was to determine the importance of the sodium-glucose cotransporter SGLT1 and the glucose uniporter GLUT2 in intestinal glucose absorption during oral glucose tolerance tests (OGTTs) in mice. Glucose absorption was determined in mice using positron emission tomography and three non-metabolizable glucose probes: one specific for SGLTs, one specific for GLUTs, and one a substrate for both SGLTs and GLUTs. Absorption was determined in wild-type, Sglt1-/- and Glut2-/- mice. Gastric emptying was a rate-limiting step in absorption. SGLT1, but not GLUT2, was important in fast glucose absorption. In the absence of SGLT1 or GLUT2, the oral glucose load delivered to the small intestine was slowly absorbed. Oral phlorizin only inhibited the fast component of glucose absorption, but it contributed to decreasing blood glucose levels by inhibiting renal reabsorption. ABSTRACT: The current model of intestinal absorption is that SGLT1 is responsible for transport of glucose from the lumen into enterocytes across the brush border membrane, and GLUT2 for the downhill transport from the epithelium into blood across the basolateral membrane. Nevertheless, questions remain about the importance of these transporters in vivo. To address these questions, we have developed a non-invasive imaging method, positron emission tomography (PET), to monitor intestinal absorption of three non-metabolized glucose tracers during standard oral glucose tolerance tests (OGTTs) in mice. One tracer is specific for SGLTs (α-methyl-4-[18 F]fluoro-4-deoxy-d-glucopyranoside; Me-4FDG), one is specific for GLUTs (2-deoxy-2-[18 F]fluoro-d-glucose; 2-FDG), and one is a substrate for both SGLTs and GLUTs (4-deoxy-4-[18 F]fluoro-d-glucose; 4-FDG). OGTTs were conducted on adult wild-type, Sglt1-/- and Glut2-/- mice. In conscious mice, OGTTs resulted in the predictable increase in blood glucose that was blocked by phlorizin in both wild-type and Glut2-/- animals. The blood activity of both Me-4FDG and 4-FDG, but not 2-FDG, accompanied the changes in glucose concentration. PET imaging during OGTTs further shows that: (i) intestinal absorption of the glucose load depends on gastric emptying; (ii) SGLT1 is important for the fast absorption; (iii) GLUT2 is not important in absorption; and (iv) oral phlorizin reduces absorption by SGLT1, but is absorbed and blocks glucose reabsorption in the kidney. We conclude that in standard OGTTs in mice, SGLT1 is essential in fast absorption, GLUT2 does not play a significant role, and in the absence of SGLT1 the total load of glucose is slowly absorbed.

Conformational transitions of the sodium-dependent sugar transporter, vSGLT.

Sodium-dependent transporters couple the flow of Na+ ions down their electrochemical potential gradient to the uphill transport of various ligands. Many of these transporters share a common core structure composed of a five-helix inverted repeat and deliver their cargo utilizing an alternating-access mechanism. A detailed characterization of inward-facing conformations of the Na+-dependent sugar transporter from Vibrio parahaemolyticus (vSGLT) has previously been reported, but structural details on additional conformations and on how Na+ and ligand influence the equilibrium between other states remains unknown. Here, double electron-electron resonance spectroscopy, structural modeling, and molecular dynamics are utilized to deduce ligand-dependent equilibria shifts of vSGLT in micelles. In the absence and presence of saturating amounts of Na+, vSGLT favors an inward-facing conformation. Upon binding both Na+ and sugar, the equilibrium shifts toward either an outward-facing or occluded conformation. While Na+ alone does not stabilize the outward-facing state, gating charge calculations together with a kinetic model of transport suggest that the resting negative membrane potential of the cell, absent in detergent-solubilized samples, may stabilize vSGLT in an outward-open conformation where it is poised for binding external sugars. In total, these findings provide insights into ligand-induced conformational selection and delineate the transport cycle of vSGLT.

Positron emission tomography of sodium glucose cotransport activity in high grade astrocytomas.

A novel glucose transporter, the sodium glucose cotransporter 2 (SGLT2), has been demonstrated to contribute to the demand for glucose by pancreatic and prostate tumors, and its functional activity has been imaged using a SGLT specific PET imaging probe, α-methyl-4-[F-18]fluoro-4-deoxy-D-glucopyaranoside (Me-4FDG). In this study, Me-4FDG PET was extended to evaluate patients with high-grade astrocytic tumors. Me-4FDG PET scans were performed in four patients diagnosed with WHO Grade III or IV astrocytomas and control subjects, and compared with 2-deoxy-2-[F-18]fluoro-D-glucose (2-FDG) PET and magnetic resonance imaging (MRI) of the same subjects. Immunocytochemistry was carried out on Grade IV astrocytomas to determine the cellular location of SGLT proteins within the tumors. Me-4FDG retention was pronounced in astrocytomas in dramatic contrast to the lack of uptake into the normal brain, resulting in a high signal-to-noise ratio. Macroscopically, the distribution of Me-4FDG within the tumors overlapped with that of 2-FDG uptake and tumor definition using contrast-enhanced MRI images. Microscopically, the SGLT2 protein was found to be expressed in neoplastic glioblastoma cells and endothelial cells of the proliferating microvasculature. This preliminary study shows that Me-4FDG is a highly sensitive probe for visualization of high-grade astrocytomas by PET. The distribution of Me-4FDG within tumors overlapped that for 2-FDG, but the absence of background brain Me-4FDG resulted in superior imaging sensitivity. Furthermore, the presence of SGLT2 protein in astrocytoma cells and the proliferating microvasculature may offer a novel therapy using the SGLT2 inhibitors already approved by the FDA to treat type 2 diabetes mellitus.

Active site voltage clamp fluorometry of the sodium glucose cotransporter hSGLT1.

In the human sodium glucose cotransporter (hSGLT1) cycle, the protein undergoes conformational changes where the sugar-binding site alternatively faces the external and internal surfaces. Functional site-directed fluorometry was used to probe the conformational changes at the sugar-binding site. Residues (Y290, T287, H83, and N78) were mutated to cysteines. The mutants were expressed in Xenopus laevis oocytes and tagged with environmentally sensitive fluorescent rhodamines [e.g., tetramethylrhodamine (TMR)-thiols]. The fluorescence intensity was recorded as the mutants were driven into different conformations using voltage jumps. Sugar binding and transport by the fluorophore-tagged mutants were blocked, but Na+ binding and the voltage-dependent conformational transitions were unaffected. Structural models indicated that external Na+ binding opened a large aqueous vestibule (600 Å3) leading to the sugar-binding site. The fluorescence of TMR covalently linked to Y290C, T287C, and H83C decreased as the mutant proteins were driven from the inward to the outward open Na+-bound conformation. The time courses of fluorescence changes (milliseconds) were close to the SGLT1 capacitive charge movements. The quench in rhodamine fluorescence indicated that the environment of the chromophores became more polar with opening of the external gates as the protein transitioned from the inward to outward facing state. Structural analyses showed an increase in polar side chains and a decrease in hydrophobic side chains lining the vestibule, and this was reflected in solvation of the chromophore. The results demonstrate the opening and closing of external gates in real time, with the accompanying changes of polarity of the sugar vestibule.

Novel and Unexpected Functions of SGLTs.

It has been 30 years since the intestinal sodium glucose cotransporter SGLT1 was cloned, and, in the intervening years, there have been many advances that have influenced physiology and medicine. Among the first was that SGLT1 is the founding member of the human gene family SLC5, containing 11 diverse transporters and a glucose sensor. Equally surprising was that SGLTs are members of a structural family of cotransporters and exchangers in different gene families. This led to the conclusion that these proteins operate by a mechanism where transport involves the opening and closing of external and internal gates. The mechanism is shared by a wide variety of transporters in different structural families, e.g., the human facilitated glucose transporters (SLC2) in the huge major facilitator superfamily (MFS). Not surprising is the finding that mutations in Sglt genes cause the rare diseases glucose-galactose-malabsorption (GGM) and familial renal glucosuria (FRG). However, it was not envisaged that SGLT inhibitors would be used to treat diabetes mellitus, and these drugs may be able to treat cancer. Finally, in 2017, we have just learned that SGLT1 may be required to resist infection and to avoid recurrent pregnancy loss.

Dapagliflozin Binds Specifically to Sodium-Glucose Cotransporter 2 in the Proximal Renal Tubule.

Kidneys contribute to glucose homeostasis by reabsorbing filtered glucose in the proximal tubules via sodium-glucose cotransporters (SGLTs). Reabsorption is primarily handled by SGLT2, and SGLT2-specific inhibitors, including dapagliflozin, canagliflozin, and empagliflozin, increase glucose excretion and lower blood glucose levels. To resolve unanswered questions about these inhibitors, we developed a novel approach to map the distribution of functional SGLT2 proteins in rodents using positron emission tomography with 4-[18F]fluoro-dapagliflozin (F-Dapa). We detected prominent binding of intravenously injected F-Dapa in the kidney cortexes of rats and wild-type and Sglt1-knockout mice but not Sglt2-knockout mice, and injection of SGLT2 inhibitors prevented this binding. Furthermore, imaging revealed only low levels of F-Dapa in the urinary bladder, even after displacement of kidney binding with dapagliflozin. Microscopic ex vitro autoradiography of kidney showed F-Dapa binding to the apical surface of early proximal tubules. Notably, in vivo imaging did not show measureable specific binding of F-Dapa in heart, muscle, salivary glands, liver, or brain. We propose that F-Dapa is freely filtered by the kidney, binds to SGLT2 in the apical membranes of the early proximal tubule, and is subsequently reabsorbed into blood. The high density of functional SGLT2 transporters detected in the apical membrane of the proximal tubule but not detected in other organs likely accounts for the high kidney specificity of SGLT2 inhibitors. Overall, these data are consistent with data from clinical studies on SGLT2 inhibitors and provide a rationale for the mode of action of these drugs.

Structural and functional significance of water permeation through cotransporters.

Membrane transporters, in addition to their major role as specific carriers for ions and small molecules, can also behave as water channels. However, neither the location of the water pathway in the protein nor their functional importance is known. Here, we map the pathway for water and urea through the intestinal sodium/glucose cotransporter SGLT1. Molecular dynamics simulations using the atomic structure of the bacterial transporter vSGLT suggest that water permeates the same path as Na+ and sugar. On a structural model of SGLT1, based on the homology structure of vSGLT, we identified and mutated residues lining the sugar transport pathway to cysteine. The mutants were expressed in Xenopus oocytes, and the unitary water and urea permeabilities were determined before and after modifying the cysteine side chain with reversible methanethiosulfonate reagents. The results demonstrate that water and urea follow the sugar transport pathway through SGLT1. The changes in permeability, increases or decreases, with side-chain modifications depend on the location of the mutation in the region of external or internal gates, or the sugar binding site. These changes in permeability are hypothesized to be due to alterations in steric hindrance to water and urea, and/or changes in protein folding caused by mismatching of side chains in the water pathway. Water permeation through SGLT1 and other transporters bears directly on the structural mechanism for the transport of polar solutes through these proteins. Finally, in vitro experiments on mouse small intestine show that SGLT1 accounts for two-thirds of the passive water flow across the gut.

Stochastic steps in secondary active sugar transport.

Secondary active transporters, such as those that adopt the leucine-transporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-facing state of the transporter. Our simulations reveal that the order of release is stochastic. Functional experiments were designed to test this prediction on the human homolog, hSGLT1, and we also found that cytoplasmic release is not ordered, but we confirmed that substrate and ion binding from the extracellular space is ordered. Our findings unify conflicting published results concerning cytoplasmic release of ions and substrate and hint at the possibility that other transporters in the superfamily may lack coordination between ions and substrate in the inward-facing state.

Revisiting the physiological roles of SGLTs and GLUTs using positron emission tomography in mice.

KEY POINTS: Glucose transporters are central players in glucose homeostasis. There are two major classes of glucose transporters in the body, the passive facilitative glucose transporters (GLUTs) and the secondary active sodium-coupled glucose transporters (SGLTs). In the present study, we report the use of a non-invasive imaging technique, positron emission tomography, in mice aiming to evaluate the role of GLUTs and SGLTs in controlling glucose distribution and utilization. We show that GLUTs are most significant for glucose uptake into the brain and liver, whereas SGLTs are important in glucose recovery in the kidney. This work provides further support for the use of SGLT imaging in the investigation of the role of SGLT transporters in human physiology and diseases such as diabetes and cancer. ABSTRACT: The importance of sodium-coupled glucose transporters (SGLTs) and facilitative glucose transporters (GLUTs) in glucose homeostasis was studied in mice using fluorine-18 labelled glucose molecular imaging probes and non-invasive positron emission tomography (PET) imaging. The probes were: α-methyl-4-[F-18]-fluoro-4-deoxy-d-glucopyranoside (Me-4FDG), a substrate for SGLTs; 4-deoxy-4-[F-18]-fluoro-d-glucose (4-FDG), a substrate for SGLTs and GLUTs; and 2-deoxy-2-[F-18]-fluoro-d-glucose (2-FDG), a substrate for GLUTs. These radiolabelled imaging probes were injected i.v. into wild-type, Sglt1(-/-) , Sglt2(-/-) and Glut2(-/-) mice and their dynamic whole-body distribution was determined using microPET. The distribution of 2-FDG was similar to that reported earlier (i.e. it accumulated in the brain, heart, liver and kidney, and was excreted into the urinary bladder). There was little change in the distribution of 2-FDG in Glut2(-/-) mice, apart from a reduction in the rate of uptake into liver. The major differences between Me-4FDG and 2-FDG were that Me-4FDG did not enter the brain and was not excreted into the urinary bladder. There was urinary excretion of Me-4FDG in Sglt1(-/-) and Sglt2(-/-) mice. However, Me-4FDG was not reabsorbed in the kidney in Glut2(-/-) mice. There were no differences in Me-4FDG uptake into the heart of wild-type, Sglt1(-/-) and Sglt2(-/-) mice. We conclude that GLUT2 is important in glucose liver transport and reabsorption of glucose in the kidney along with SGLT2 and SGLT1. Complete reabsorption of Me-4FDG from the glomerular filtrate in wild-type mice and the absence of reabsorption in the kidney in Glut2(-/-) mice confirm the importance of GLUT2 in glucose absorption across the proximal tubule.

Functional expression of sodium-glucose transporters in cancer.

Glucose is a major metabolic substrate required for cancer cell survival and growth. It is mainly imported into cells by facilitated glucose transporters (GLUTs). Here we demonstrate the importance of another glucose import system, the sodium-dependent glucose transporters (SGLTs), in pancreatic and prostate adenocarcinomas, and investigate their role in cancer cell survival. Three experimental approaches were used: (i) immunohistochemical mapping of SGLT1 and SGLT2 distribution in tumors; (ii) measurement of glucose uptake in fresh isolated tumors using an SGLT-specific radioactive glucose analog, α-methyl-4-deoxy-4-[(18)F]fluoro-D-glucopyranoside (Me4FDG), which is not transported by GLUTs; and (iii) measurement of in vivo SGLT activity in mouse models of pancreatic and prostate cancer using Me4FDG-PET imaging. We found that SGLT2 is functionally expressed in pancreatic and prostate adenocarcinomas, and provide evidence that SGLT2 inhibitors block glucose uptake and reduce tumor growth and survival in a xenograft model of pancreatic cancer. We suggest that Me4FDG-PET imaging may be used to diagnose and stage pancreatic and prostate cancers, and that SGLT2 inhibitors, currently in use for treating diabetes, may be useful for cancer therapy.