There are two classes of glucose transporters involved in glucose homeostasis in the body, the facilitated transporters or uniporters (GLUTs) and the active transporters or symporters (SGLTs). The energy for active glucose transport is provided by the sodium gradient across the cell membrane, the Na(+) glucose cotransport hypothesis first proposed in 1960 by Crane. Since the cloning of SGLT1 in 1987, there have been advances in the genetics, molecular biology, biochemistry, biophysics, and structure of SGLTs. There are 12 members of the human SGLT (SLC5) gene family, including cotransporters for sugars, anions, vitamins, and short-chain fatty acids. Here we give a personal review of these advances. The SGLTs belong to a structural class of membrane proteins from unrelated gene families of antiporters and Na(+) and H(+) symporters. This class shares a common atomic architecture and a common transport mechanism. SGLTs also function as water and urea channels, glucose sensors, and coupled-water and urea transporters. We also discuss the physiology and pathophysiology of SGLTs, e.g., glucose galactose malabsorption and familial renal glycosuria, and briefly report on targeting of SGLTs for new therapies for diabetes.

Membrane transporters that use energy stored in sodium gradients to drive nutrients into cells constitute a major class of proteins. We report the crystal structure of a member of the solute sodium symporters (SSS), the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT). The ~3.0Å structure contains 14 transmembrane helices in an inward facing conformation with a core structure of inverted repeats of 5 TM helices (TM2-TM6 and TM7-TM11). Galactose is bound in the center of the core, occluded from the outside solutions by hydrophobic residues. Surprisingly, the architecture of the core is similar to the leucine transporter (LeuT) from a different gene family. Modeling the outward-facing conformation based on the LeuT structure, in conjunction with biophysical data, provides insight into structural rearrangements for active transport.

Secondary active glucose transport occurs by at least four members of the SLC5 gene family. This review considers the structure and function of two premier members, SGLT1 and SGLT2, and their role in intestinal glucose absorption and renal glucose reabsorption. Genetics disorders of SGLTs include GlucoseGalactose Malabsorption, and Familial Renal Glucosuria. SGLT1 plays a central role in Oral Rehydration Therapy used so effectively to treat secretory diarrhoea such as cholera. Increasing attention is being focused on SGLTs as drug targets for the therapy of diabetes.

We have examined the expression and function of a previously undescribed human member (SGLT3͞SLC5A4) of the sodium͞glu-cose cotransporter gene family (SLC5) that was first identified by the chromosome 22 genome project. The cDNA was cloned and sequenced, confirming that the gene coded for a 659-residue protein with 70% amino acid identity to the human SGLT1. RT-PCR and Western blotting showed that the gene was transcribed and mRNA was translated in human skeletal muscle and small intestine. Immunofluorescence microscopy indicated that in the small intestine the protein was expressed in cholinergic neurons in the submucosal and myenteric plexuses, but not in enterocytes. In skeletal muscle SGLT3 immunoreactivity colocalized with the nicotinic acetylcholine receptor. Functional studies using the Xenopus laevis oocyte expression system showed that hSGLT3 was incapable of sugar transport, even though SGLT3 was efficiently inserted into the plasma membrane. Electrophysiological assays revealed that glucose caused a specific, phlorizin-sensitive, Na ؉ -dependent depolarization of the membrane potential. Uptake assays under voltage clamp showed that the glucose-induced inward currents were not accompanied by glucose transport. We suggest that SGLT3 is not a Na ؉ ͞glucose cotransporter but instead a glucose sensor in the plasma membrane of cholinergic neurons, skeletal muscle, and other tissues. This points to an unexpected role of glucose and SLC5 proteins in physiology, and highlights the importance of determining the tissue expression and function of new members of gene families.Na͞sugar cotransporter ͉ human SGLT3 ͉ muscle

The human Na(+)/D-glucose cotransporter 2 (hSGLT2) is believed to be responsible for the bulk of glucose reabsorption in the kidney proximal convoluted tubule. Since blocking reabsorption increases urinary glucose excretion, hSGLT2 has become a novel drug target for Type 2 diabetes treatment. Glucose transport by hSGLT2 was studied at 37°C in human embryonic kidney 293T cells using whole cell patch-clamp electrophysiology. We compared hSGLT2 with hSGLT1, the transporter in the straight proximal tubule (S3 segment). hSGLT2 transports with surprisingly similar glucose affinity and lower concentrative power than hSGLT1: Na(+)/D-glucose cotransport by hSGLT2 was electrogenic with apparent glucose and Na(+) affinities of 5 and 25 mM, and a Na(+):glucose coupling ratio of 1; hSGLT1 affinities were 2 and 70 mM and coupling ratio of 2. Both proteins showed voltage-dependent steady-state transport; however, unlike hSGLT1, hSGLT2 did not exhibit detectable pre-steady-state currents in response to rapid jumps in membrane voltage. D-Galactose was transported by both proteins, but with very low affinity by hSGLT2 (≥100 vs. 6 mM). β-D-Glucopyranosides were either substrates or blockers. Phlorizin exhibited higher affinity with hSGLT2 (K(i) 11 vs. 140 nM) and a lower Off-rate (0.03 vs. 0.2 s⁻¹) compared with hSGLT1. These studies indicate that, in the early proximal tubule, hSGLT2 works at 50% capacity and becomes saturated only when glucose is ≥35 mM. Furthermore, results on hSGLT1 suggest it may play a significant role in the reabsorption of filtered glucose in the late proximal tubule. Our electrophysiological study provides groundwork for a molecular understanding of how hSGLT inhibitors affect renal glucose reabsorption.

The mechanism by which cotransport proteins couple their substrates across cell membranes is not known. A commonly proposed model is that cotransport results from ligand-induced conformational transitions that change the accessibility of ligand-binding sites from one side of the membrane to the other. To test this model, we have measured the accessibility of covalent probes to a cysteine residue (Q457C) placed in the putative sugar-translocation domain of the Na ؉ ͞glucose cotransporter (SGLT1). The mutant protein Q457C was able to transport sugar, but transport was abolished after alkylation by methanethiosulfonate reagents. Alkylation blocked sugar translocation but not sugar binding. Accessibility of Q457C to alkylating reagents required external Na ؉ and was blocked by external sugar and phlorizin. The voltage dependence of accessibility was directly correlated with the presteady-state charge movement of SGLT1. Voltage-jump experiments with rhodamine-6-maleimide-labeled Q457C showed that the time course and level of changes in f luorescence closely followed the presteadystate charge movement. We conclude that conformational changes are responsible for the coupling of Na ؉ and sugar transport and that Q457 plays a critical role in sugar translocation by SGLT1.Cotransporters are a major class of membrane proteins that are formed by members of several gene families. They share the common property of being able to couple the electrochemical potential gradient of a cation (Na ϩ or H ϩ ) to transport of organic solutes, ions, and water uphill into cells (see refs. 1 and 2). Although the mechanism of energy transduction is unknown, cotransporters share several common functional properties. For example, they exhibit presteady-state currents with step changes in membrane potential, which suggests the existence of a common mechanism.Kinetic models of cotransport have been proposed. Most popular are: alternating access models in which binding of multiple substrates at distinct sites that are only accessible on one side of the membrane at a time; transport then occurs via ligand and voltage induced conformational changes (3, 4); and channel-like models with multiple substrate occupancy without conformational changes (5). Mathematical simulations of each type of model can account for many experimental observations. In the alternating access model, the presteadystate currents are caused by both relaxations of charged or polar residues in the protein in response to voltage perturbations and movement of the transported ions in the membrane field (4, 6). In contrast, in the channel model, presteady-state currents are strictly caused by the transported ions (5).The advent of cysteine mutagenesis and derivatization with probe reagents has proved to be a useful tool for structure͞ function studies of ion channels and transporters (7-9), and membrane voltage has been found to affect the accessibility of residues in the transmembrane domain (8, 10). The dependence of cotransport function on both substrates and membrane vo...

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-, 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 | pancreatic cancer | prostate cancer | SGLT2-inhibitors P ancreatic cancer is the fourth-leading cause of cancer-related death in the United States (behind only lung, colon, and breast cancers), with 46,420 estimated new cases in 2014 and a mortality that almost equals incidence (39,590 estimated deaths in 2014); the overall 5-y survival rate is only 7% (seer.cancer.gov/statfacts/html/ pancreas.html). Prostate cancer is the most frequent cancer in men in the United States, with 233,000 estimated new cases in 2014. Despite widespread adoption of screening programs, prostate cancer is still the second-leading cause of cancer-related death in men, second only to lung cancer (seer.cancer.gov/statfacts/html/prost.

SUMMARY1. The ontogenic development of the intestinal Na+-glucose co-transporter was measured in lambs as a function of diet. Transport activity was assayed in brushborder membrane vesicles and the expression of transport protein in the brushborder membrane determined by Western analysis.2. Na+-dependent D-glucose transport increased to a maximum (300-700 pmol mg' s') within the first 2 weeks of birth and then declined to negligible amounts (< 10 pmol mg-' s-1) over the next 8 weeks. There was no further change over the next 2-3 years. Early changes were associated with modifications in both the maximum velocity Vma. for transport and expression of carrier protein in the brush-border plasma membrane.3. Maintaining lambs on a milk replacer diet beyond the normal weaning period prevented the normal decline in the expression of Na+-glucose co-transport. At 5 weeks the transport rate was 433 + 150 pmol mg-' s-I in lambs maintained on milk replacer, but only 79 + 40 pmol mg-' s-I in normally reared control lambs.4. Infusing the proximal intestine of 2-to 3-year-old sheep with 30 mM-D-glucose for four days increased the rate of transport 40-to 80-fold above that found in control animals perfused with mannitol. A similar but smaller increase was observed in one animal perfused with the non-metabolizable sugar a-methyl-D-glucopyranoside. The induced increase in glucose transport was correlated with the expression of the co-transporter protein in the brush-border plasma membrane.5. It is concluded that the age-related decline in Na+-glucose co-transport in the sheep intestine is directly due to the decrease in D-glucose (and D-galactose) reaching the small intestine after development of the rumen. These results further suggest that luminal sugar substrates for the co-transporter promote both the maintenance and the up-regulation of the brush-border transport protein and it is the intact sugar itself which controls gene expression during enterocyte maturation. MS 8905 S. P. SHIRAZI-BEECHEY AND OTHERS