**2.3. Structural features & substrate specificities**

SGLT family members 1-6 contain between 596-681 amino acids with a 50-70% identity (67-84% similarity) where divergence in sequences can be mainly attributed to the N-and C-terminal domains of the proteins. Alternative splicing has been described for SGLT4-6, however, whether respective functional proteins with varying amino acid composition are encoded has yet to be shown. A common structural component among the large gene family of sodium:sol‐ ute symporters (SSF) is the presence of a consensus pattern (Figure 2A). The consensus sequence for the six SGLTs and for SMIT1 is located near the N-terminal domain of the proteins (Figure 2A).

A secondary structural model for human SGLT1 predicts the presence of 14 transmembrane helices. The model is based on N-glycosylation and cysteine scanning mutagenesis, antibody tagging, mass spectrometry as well as computer algorithms predicting membrane-spanning regions (Figure 2A). Freeze-fracture electron microscopy provided direct evidence that both SGLT1 and vSGLT (from *Vibrio parahaemolyticus*) function as 14 transmembrane helical monomers. he recent crystal structure for the sodium/galactose symporter vSGLT demon‐ strated the presence of 14 transmembrane helices providing evidence for the secondary structural model of human SGLT1.

SGLT are highly glycosylated membrane proteins for example SGLT1 contains a N-linked glycosylation site at position N248. However, for SGLT1, glycosylation appears not to be required for functional expression, indicating proper folding and membrane targeting in its absence.

The transport kinetics and substrate specificities have been intensively studied for SGLT1, 2, 3, and more recently 4, mainly based on electrophysiological and biochemical studies upon heterologous expression of the transporters in *Xenopus laevis* oocytes. SGLT1-4 and SMIT1 and 2 all transport (or bind in the case of human SGLT3) D-glucose and the non-metabolizable alpha-methyl-d-glucopyranoside (α-DMG). Transport of those substrates is inhibited by the glycoside phlorizin [6].

SGLT are only able to transport sugars with a pyranose ring, cyclic polyhydroxy alcohols are not transported. The importance of the single hydroxyl groups for substrate recognition has been well characterized. The oxygen is essential for transport by human SGLT1-while sulfur substitution lowers affinity, nitrogen is not tolerated. This particular feature does not apply to SGLT3 for which imino sugars-containing an amine group in place of a hydroxyl group-are ligands. Based on mutational analysis of SGLTs and the crystal structure of vSGLT, which is 32% identical to human SGLT1, residues that coordinate substrate recognition have been shown to be relatively conserved. An exception to that is the human SGLT3, a glucose sensor, which can be converted to a functional transporter based on a single amino acid exchange.

In addition to glucose and other monosaccharides, SGLTs also transport glycosides. Those can be either substrates such as indican and arbutin, or actual inhibitors such as the highly potent, classic competitive SGLT inhibitor phlorizin, a naturally occurring β-glucoside (see below).

The ability to recognize galactose as substrate by SLC5 family members has been attributed to the presence of a threonine corresponding to amino acid 460 in human SGLT1.

Ion selectivity and stoichiometry has been well characterized for SGLTs. The transporters are selective for the cotransport cation Na+ (Km=4mM). While Li<sup>+</sup> (Km=9mM) and H<sup>+</sup> (Km=7µM) can replace Na+, no other monovalent cation is accepted. The Na+ to glucose transport stoichiometry is established for SGLT1-3, where two Na+ ions bind to SGLT1 and 3, and only one Na+ is required to drive SGLT2 activity. Despite crystallographic information for vSGLT, the electron density is not sufficient to assign binding sites for small single ions such as sodium. However, using mutational analysis and superimposition of structural models from the solute symport‐ ers vSGLT, LeuT, and Mhp1, a sodium binding site for vSGLT is suggested to be close to the sugar binding residues in transmembrane domains 1 and 8. The predicted cation binding site in vSGLT appears to allow accessibility to the cytoplasmic aqueous phase.

The mechanism of sodium-driven glucose transport has been intensively investigated for SGLT1, applying various methodologies that allow the kinetics of transport to be deter‐ mined using heterologous expression of the transporter in *Xenopus laevis* oocytes. From the kinetic measurements a 6-state equilibrium model is proposed, where conformational changes dependent on cation and sugar binding, transport and cytoplasmic release are integrated. The six kinetic states describe the "empty" transporter, the sodium bound form, and the sodium and glucose bound transporter at the external and internal plasma membrane surfaces (Figure 5A).

SGLT1 mediated glucose transport has been characterized regarding its kinetics, conforma‐ tional changes and the significance of residues for substrate/inhibitor binding. However, many questions remain unanswered such as the precise identity of the second-sodium binding site for SGLT1, and the location of the phlorizin binding site in SGLT1 and SGLT2, which may be of relevance for SGLT2 selective inhibitors that, recently approved, represent a new treatment option for Type 2 diabetes mellitus (T2DM) (see below).

## **2.4. SGLT1 (***SLC5A1)*

More distant relatives of the *SLC5A* gene family are the iodide transporters NIS (sodium-iodide symporter [SLC5A5]) and AIT (apical iodide transporter [SLC5A11]), the Na+/Cl-/choline transporter (CHT, [SLC5A7]) and the sodium-dependent multivitamin transporter (SMVT, [SLC5A6]). NIS and AIT are expressed in the thyroid gland. While NIS is responsible for iodide uptake which is required for production of T3 and T4, AIT is thought to catalyse the movement of iodide from the thyrocyte cytoplasm to the lumen of the gland. SMVT is widely expressed,

CHT is mainly found in the central nervous system. Biochemically, the CHT mediates Na+/

SGLT family members 1-6 contain between 596-681 amino acids with a 50-70% identity (67-84% similarity) where divergence in sequences can be mainly attributed to the N-and C-terminal domains of the proteins. Alternative splicing has been described for SGLT4-6, however, whether respective functional proteins with varying amino acid composition are encoded has yet to be shown. A common structural component among the large gene family of sodium:sol‐ ute symporters (SSF) is the presence of a consensus pattern (Figure 2A). The consensus sequence for the six SGLTs and for SMIT1 is located near the N-terminal domain of the proteins

A secondary structural model for human SGLT1 predicts the presence of 14 transmembrane helices. The model is based on N-glycosylation and cysteine scanning mutagenesis, antibody tagging, mass spectrometry as well as computer algorithms predicting membrane-spanning regions (Figure 2A). Freeze-fracture electron microscopy provided direct evidence that both SGLT1 and vSGLT (from *Vibrio parahaemolyticus*) function as 14 transmembrane helical monomers. he recent crystal structure for the sodium/galactose symporter vSGLT demon‐ strated the presence of 14 transmembrane helices providing evidence for the secondary

SGLT are highly glycosylated membrane proteins for example SGLT1 contains a N-linked glycosylation site at position N248. However, for SGLT1, glycosylation appears not to be required for functional expression, indicating proper folding and membrane targeting in its

The transport kinetics and substrate specificities have been intensively studied for SGLT1, 2, 3, and more recently 4, mainly based on electrophysiological and biochemical studies upon heterologous expression of the transporters in *Xenopus laevis* oocytes. SGLT1-4 and SMIT1 and 2 all transport (or bind in the case of human SGLT3) D-glucose and the non-metabolizable alpha-methyl-d-glucopyranoside (α-DMG). Transport of those substrates is inhibited by the

SGLT are only able to transport sugars with a pyranose ring, cyclic polyhydroxy alcohols are not transported. The importance of the single hydroxyl groups for substrate recognition has been well characterized. The oxygen is essential for transport by human SGLT1-while sulfur substitution lowers affinity, nitrogen is not tolerated. This particular feature does not apply to

choline co-transport in a chloride dependent manner.

**2.3. Structural features & substrate specificities**

while

10 Glucose Homeostasis

(Figure 2A).

absence.

glycoside phlorizin [6].

structural model of human SGLT1.

In 1987, the laboratory of Ernest Wright cloned the first sodium-dependent glucose transporter from rabbit intestinal mRNA by an expressing cloning strategy using *Xenopus laevis* oocytes [1]. SGLT1 is primarily expressed in the brush border membrane of mature enterocytes in the small intestine and catalyses the absorption of the dietary sugars glucose and galactose from the gut lumen. SGLT1 is also expressed in the kidney on the luminal surface of cells within the S3 segment of the proximal tubule, where it contributes to renal glucose reabsorption (Figure 3A).

FRG is an autosomal recessive disorder and is diagnosed by persistent isolated glucosuria (urine excretion >1 g/day) with normal fasting plasma glucose levels and oral glucose tolerance.

Mammalian Sugar Transporters http://dx.doi.org/10.5772/58325 13

Since the first SLC5A2 mutation in FRG was presented in 2002 [9], forty-four mutations have been identified including premature stops, frame shifts, and missense mutations. Although the pattern of inheritance for FRG is of co-dominance, a clear definition of genotype-phenotype correlation has not been established. Individuals with similar or even identical mutations display a broad range of severity in glucosuria, indicating that environmental, as well as genetic, factors affect urinary glucose reabsorption. Since, thus far, none of the FRG mutations has been tested for functional SGLT2 effects it is unknown how the various mutations relate

FRG established the fundamental role of SGLT2 in renal glucose reabsorption. Since patients with FRG are not affected by severe clinical consequences, it is considered a benign condition,

The metabolic consequences of SGLT2 deficiency in mice have been investigated in a model of diet-induced obesity and associated insulin resistance and a genetic model of T2DM, the db/db mouse strain [10]. Deletion of SGLT2 leads to increased urine output and a tremendous increase in glucosuria that is associated with compensatory increases in feeding, drinking, and activity. SGLT2 knockout mice are protected from diet-induced hyperglycemia and glucose intolerance and have reduced plasma insulin concentrations. In the diabetic db/db mouse, deficiency of SGLT2 prevents fasting hyperglycemia and is associated with normalized plasma insulin levels and preserved pancreatic β-cell function. These data confirm the concept of glucotoxicity which was established by studying the anti-diabetic effects of blocking renal glucose reabsorption in diabetic rats by pharmacological means of SGLT inhibition using

T2DM is characterized by hyperglycemia that results both from peripheral resistance to the action of insulin and from progressive failure of the pancreatic β-cell to compensate for the increasing demand for insulin. Chronic hyperglycemia triggers glucotoxicity, a term summarizing the vicious cycle between hyperglycemia inducing β-cell dysfunction and insulin resistance that aggravates disease progression leading to micro-and macrovascular

Current treatments for T2DM come with significant limitations regarding their potential to induce adverse effects. Metformin can cause gastrointestinal effects such as diarrhea and nausea, while sulfonylureas and insulin can induce hypoglycemia and are associated with weight gain. Thiazolidinediones that act as insulin sensitizers can induce weight gain, and are associated with edema and are potentially associated with an increased cardiovascular risk. GLP-1 analogues which are incretin mimicking agents can cause nausea and diarrhea. New therapeutic strategies are needed that are not only effective in terms of glucose control, but

*2.5.2. SGLT2 inhibitors – A new concept for the treatment of Type 2 diabetes*

to the severity of glucosuria.

more a phenotype than a disease.

*Mouse models of SGLT2 deficiency*

phlorizin [11].

complications.

SGLT1 is a high-affinity, low-capacity transporter with a Km of 0.5 mM for the substrate αMDG in *Xenopus laevis* oocytes. Substrate transport of glucose is coupled to symport of two sodium ions. The protein is highly glycosylated which leads to an apparent molecular weight of 75 kDa.

## *2.4.1. SGLT1 physiology*
