Chiara Ghezzi

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.

Functional expression of sodium-glucose transporters in cancer

Significance Cancers require high amounts of glucose to grow and survive, and dogma is that uptake is facilitated by passive glucose transporters (GLUTs). We have identified a new mechanism to import glucose into pancreatic and prostate cancer cells, namely active glucose transport mediated by sodium-dependent glucose transporters (SGLTs). This means that the specific radioactive imaging probe for SGLTs, α-methyl-4-deoxy-4-[18F]fluoro-d-glucopyranoside, may be used along with positron-emission tomography to diagnose and stage pancreatic and prostate cancers, tumors in which the GLUT probe 2-[18F]fluoro-2-deoxy-d-glucose has questionable utility. Moreover, we suggest, based on our results in mouse models, that Food and Drug Administration-approved SGLT2 inhibitors may be used to reduce the viability of pancreatic and prostate cancer cells in patients. 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-[18F]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.

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.

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

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.

Stochastic steps in secondary active sugar transport

Significance The potential energy stored in ion gradients across cell membranes drives nutrients in and out of cells by cotransport proteins, e.g., uphill glucose accumulation in cells by sodium cotransporters. Insight into the mechanism of cotransport has been obtained from high-resolution atomic structures of the transporters, but further progress requires dynamic information about ion and substrate movements through the proteins. We have used multiple long molecular-dynamic simulations and electrophysiological assays to explore the dynamics of the transport cycle. Ligands bound to sodium-dependent glucose transporters are released to the cytoplasm stochastically, whereas release to the external solution is ordered with sugar first. The order of events is intimately tied to how the protein converts the energy stored in an ion gradient into a sugar gradient. 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.

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.

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.

Real‐time imaging of sodium glucose transporter (SGLT1) trafficking and activity in single cells

The processes controlling targeting of glucose transporters to apical and basolateral membranes of polarized cells are complex and not‐well understood. We have engineered SGLT1 and GLUT4 constructs linked to fluorescent proteins to highlight the differences in transporter expression and trafficking, in real time, in different cell types. Activity was assessed in parallel using a FRET glucose sensor. In COS cells and HEK cells, SGLT1 was distributed between the plasma membrane and intracellular compartments, but there was little expression in CHO cells. Trafficking was investigated using the lysosome inhibitors NH4Cl (10 mmol/L) and chloroquine (150 μmol/L) and the proteasome inhibitors MG‐262 (1 μmol/L) and lactacystin (5 μmol/L). Lysosome inhibitors caused SGLT1 accumulation into intracellular bodies, whereas proteasome inhibitors induced SGLT1 accumulation in the plasma membrane, even in CHO cells. Our data suggest that a fraction of SGLT1 is rapidly degraded by lysosomes and never reached the plasma membrane; another fraction reaches the membrane and is subsequently degraded by lysosomes following internalization. The latter process is regulated by the ubiquitin/proteasome pathway, acting at a late stage of the lysosomal pathway. Using the cholesterol inhibitor MβCD (3 mmol/L), a dominant negative dynamin (K44A) and caveolin, we showed that SGLT1 internalization is lipid raft‐mediated, but caveolin‐independent. In contrast, GLUT4 internalization is dynamin‐dependent, but cholesterol‐independent. The physiological relevance of these data is discussed in terms of differential membrane compartmentalization of the transporters and expression under stress conditions.

Fingerprints of hSGLT5 sugar and cation selectivity

Sodium glucose cotransporters (SGLTs) mediate the translocation of carbohydrates across the brush border membrane of different organs such as intestine, kidney, and brain. The human SGLT5 (hSGLT5), in particular, is localized in the kidney were it is responsible for mannose and fructose reabsorption from the glomerular filtrate as confirmed by more recent studies on hSGLT5 knockout mice. Here we characterize the functional properties of hSGLT5 expressed in a stable T-Rex-HEK-293 cell line using biochemical and electrophysiological assays. We confirmed that hSGLT5 is a sodium/mannose transporter that is blocked by phlorizin. Li(+) and H(+) ions were also able to drive mannose transport, and transport was electrogenic. Our results moreover indicate that substrates require a pyranose ring with an axial hydroxyl group (-OH) on carbon 2 (C-2). Compared with Na(+)/glucose cotransport, the level of function of Na(+)/mannose cotransport in rat kidney slices was low.

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

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.