Volume 102, Issue 1 p. 5-13
Review Article
Free Access

Putative tissue location and function of the SLC5 family member SGLT3

Matúš Soták

Corresponding Author

Matúš Soták

Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Mölndal, Sweden

Corresponding author M. Soták: AstraZeneca, Pepparedsleden 1, Mölndal 431 83, Sweden.  Email: [email protected], [email protected]Search for more papers by this author
Joanne Marks

Joanne Marks

Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK

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Robert J. Unwin

Robert J. Unwin

Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Mölndal, Sweden

Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK

Department of Physiology and Neuroscience, University of Gothenburg, Gothenburg, Sweden

Centre for Nephrology, University College London, London, UK

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First published: 11 November 2016
Citations: 27

Edited by: Kate Denton

Abstract

New Findings

  • What is the topic of this review?

    This review summarizes the evidence on the localization, electrophysiological properties, agonist specificity and putative physiological role of sodium–glucose transporter 3 (SGLT3).

  • What advances does it highlight?

    Published information is reviewed in some detail by comparing human and rodent isoforms, as well as advances in testing hypotheses for the physiological role of SGLT3 as a glucose sensor or incretin release mediator. We provide a critical overview of available published data and discuss a putative functional role for SGLT3 in human and mouse physiology.

Sodium–glucose transporter 3 (SGLT3) has attracted interest because of its putative role as a glucose sensor, rather than a sugar transporter, in contrast to its co-family members SGLT1 and SGLT2. Significant progress has been made in characterizing the electrophysiological properties in vitro of the single human SGLT3 isoform and the two mouse isoforms, SGLT3a and SGLT3b. Although early reports indicated SGLT3 expression in the small intestinal myenteric and submucosal neurones, hypothalamic neurones, portal vein and kidney, a lack of reliable antibodies has left unanswered its exact tissue and cellular localization. Several hypotheses for a role of SGLT3 in glucose sensing, gastric emptying, glucagon-like peptide-1 release and post-Roux-en-Y gastric bypass remodelling have been explored, but so far there is only limited and indirect supportive evidence using non-specific agonists/antagonists, with no firm conclusions. There are no published or available data in knockout animals, and translation is difficult because of its different isoforms in human versus rodent, as well as a lack of selective agonists or antagonists, all of which make SGLT3 challenging to study. However, its unique electrophysiological properties, ubiquitous expression at the mRNA level, enrichment in the small intestine and potential, but uncertain, physiological role demand more attention. The purpose of this overview and review of SGLT3 biology is to provide an update, highlight the gaps in our knowledge and try to signpost potential ways forward to define its likely function in vivo.

Introduction

The SLC5 family is exemplified by the SGLT1 isoform (Slc5a1), which is responsible for active glucose absorption in the intestine but is also present in the kidney proximal tubule, and the SGLT2 isoform (Slc5a2), which is almost exclusive to the renal epithelium and is responsible for the bulk of glucose reabsorption by the kidney. SGLT2 has become topical recently, because it is an effective therapeutic target for lowering blood glucose concentrations in poorly controlled diabetes mellitus. Targeting this transport pathway in the kidney has been approved by some authorities as a second-line therapy in type 2 diabetes after metformin. The advances in SGLT1 and SGLT2 biology and function have been reviewed comprehensively elsewhere (Poulsen et al. 2015; Lehmann & Hornby, 2016; Song et al. 2016), and in the present review we focus on recent developments in our understanding of the distribution and function of another SLC5 family member and putative glucose sensor, SGLT3 (Slc5a4, solute carrier family 5 member 4). Its function and topographical distribution have been less well studied and are still incompletely defined. We review its gene structure, electrophysiological and transport properties, differences between human and rodent isoforms, tissue and cellular distribution, and putative physiological role(s). Finally, we emphasize the gaps in current knowledge and outline some future perspectives.

SGLT3 (Slc5a4)

Structure

The human SLC5A4 gene is located on chromosome 22 at locus 22q12.3. The gene contains 17 exons; SLC5A1, coding SGLT1, is close by on the same chromosome. In contrast, rats and mice have two isoforms, Slc5a4a and Slc5a4b. In rodents, both isoforms are in close proximity on the same chromosome, chromosome 10 and 20 in the mouse and rat, respectively. So far, the mouse and rat are the only species known to have two isoforms of Slc5a4, making it more difficult to extrapolate findings from rodent models to man. Human SLC5A4 complementary DNA was fully cloned by Díez-Sampedro et al. (2003). In the mouse, the Slc5a4a isoform was the first to be cloned (Tabatabai et al. 2003), followed by Slc5a4b, and then rat Slc5a4a (Barcelona et al. 2012). Rat Slc5a4b has not been fully cloned, and its sequence is predicted from the genomic sequence.

The identity of the amino acid sequence of SGLT3 is ∼70% compared with SGLT1, and 55–60% compared with SGLT2 in each species (see Table 1). Interestingly, mouse and rat SGLT3a isoforms are closer (79–80%) to human SGLT3 than SGLT3b isoforms (76%). Basic phylogram analysis confirmed a closer relationship of mouse and rat SGLT3a isoforms to human SGLT3. Consistent with this, amino acid residues in the conserved ligand binding sites of rodent SGLT3a isoforms are more similar to human SGLT3 (only residue F101 in the outer gate is different; Table 2), suggesting a distinct functional role of the two rodent isoforms. Indeed, mSGLT3b is weakly able to transport glucose in a similar way to SGLT1. In contrast, mSGLT3a does not transport glucose or generate currents at pH 7.4 (Barcelona et al. 2012). The possibility that these rodent isoforms may have distinct roles is also consistent with the high level of identity between the corresponding mouse and rat SGLT3a (93%) and SGLT3b (90%) isoforms, respectively, whereas the identity between the SGLT3a and SGLT3b isoforms for mouse and rat is only ∼75%. Likewise, conservation in the ligand bindings sites is very high between rat and mouse isoforms (Table 2).

Table 1. Protein sequence identity matrix of human and rodent sodium–glucose transporter (SGLT) isoforms 1, 2 and 3
hSGLT3 mSGLT3a rSGLT3a mSGLT3b rSGLT3b hSGLT1 mSGLT1 rSGLT1 hSGLT2 mSGLT2 rSGLT2
hSGLT3 100.0 79.2 80.3 76.5 76.8 69.6 71.6 71.9 56.5 56.4 55.3
mSGLT3a 79.2 100.0 92.8 75.3 75.0 69.0 70.1 69.7 56.4 56.2 56.3
rSGLT3a 80.3 92.8 100.0 76.4 76.7 69.8 71.5 71.5 56.4 56.1 56.4
mSGLT3b 76.5 75.3 76.4 100.0 90.2 70.4 71.8 71.5 58.4 59.5 57.9
rSGLT3b 76.8 75.0 76.7 90.2 100.0 71.3 72.4 72.3 58.4 58.6 57.3
hSGLT1 69.6 69.0 69.8 70.4 71.3 100.0 88.0 87.8 58.9 60.9 58.9
mSGLT1 71.6 70.1 71.5 71.8 72.4 88.0 100.0 95.8 59.0 60.2 59.5
rSGLT1 71.9 69.7 71.5 71.5 72.3 87.8 95.8 100.0 59.3 60.6 59.8
hSGLT2 56.5 56.4 56.4 58.4 58.4 58.9 59.0 59.3 100.0 91.0 91.2
mSGLT2 56.4 56.2 56.1 59.5 58.6 60.9 60.2 60.6 91.0 100.0 90.1
rSGLT2 55.3 56.3 56.4 57.9 57.3 58.9 59.5 59.8 91.2 90.1 100.0
Table 2. A comparison of corresponding amino acid residues in putative ligand binding sites and gates
Sugar binding Na+ binding Outer gates Inner gates
H83 E102 A105 K321 T287 W291 Q457 N78 A76 I79 S389 S392 S393 L87 F101 F453 Y290
hSGLT3 H E S K A W E N A I S S S L F I Y
mSGLT3a H E S K A W E N A I S S S L F V Y
rSGLT3a H E S K A W E N A I S S S L F I Y
mSGLT3b H E A K A W G N A I S S S L V F Y
rSGLT3b H E A K A W S N A I S S S L V F Y
hSGLT1 H E A K T W Q N A I S S S L F F Y
mSGLT1 H E A K A W Q N A I S S S L F F Y
rSGLT1 H E A K A W Q N A I S S S L F F Y
hSGLT2 H E A K S W Q N A I A S S L F F Y
mSGLT2 H E A K S W Q N A I A S S L F F Y
rSGLT2 H E A K S W Q N A I A S S L F F Y
Conservation * * * * * * * * * * *
  • The conserved ligand binding sites and gates were adopted from alignment of hSGLT1 to vSGLT (Sala-Rabanal et al. 2012). The residue numbering is based on hSGLT1, and corresponding residues were obtained by multiple sequence alignment CLUSTAL O (1.2.3). *Fully conserved residue. Conservation strongly similar, scoring > 0.5 in the Gonnet PAM 250 matrix. Conservation weakly similar, scoring ≤ 0.5 in the Gonnet PAM 250 matrix. Residues in SGLT3 different from SGLT1 are in bold.

Based on in silico predictions and the solved crystal structure of the Vibrio parahaemolyticus sodium–galactose symporter (vSGLT), SGLT proteins are composed of 14 transmembrane helices (Faham et al. 2008). The structural differences between SGLT3 and other isoforms are not known, but the key amino acid in the sugar binding site has been identified. In isoforms able to transport glucose (SGLT1 and SGLT2), the residue within TM11 in the position 457 is glutamine. In contrast, SGLT3 isoforms that do not transport glucose have glutamate at this position (Table 2), except for the SGLT3b isoform of the mouse and rat, which can weakly transport glucose and has glycine or serine, respectively. Indeed, it was shown experimentally that in both hSGLT3 and mSGLT3a a change of glutamate to glutamine restored the ability of the protein to transport the glucose analogue α-methyl-d-glucose (α-MDG) in a Na+-dependent manner, and resulted in similar apparent affinities for glucose analogues to hSGLT1. Likewise, mutated hSGLT1 with glutamate at the position where glutamine is normally present loses the coupling to Na+ and has a lower affinity for glucose than wild-type hSGLT1 (Díez-Sampedro et al. 2001; Bianchi & Díez-Sampedro, 2010; Barcelona et al. 2012). The glutamine at position 457 in SGLT1 is also important for binding of the competitive SGLT inhibitor phlorizin, because mutated hSGLT3 with glutamine instead of glutamate (E457Q-hSGLT3) exhibits 100 times higher affinity for phlorizin (Bianchi & Díez-Sampedro, 2010). Interestingly, there is full conservation at the Na+ binding residues between SGLT1 and SGLT3 (Table 2). The difference in F453 in SGLT3a and hSGLT3 isoforms in the outer gate, which closes after sugar binding and makes the sugar binding site inaccessible (Sala-Rabanal et al. 2012), may be a factor in the inability of SGLT3a and hSGLT3 to transport sugars.

Tissue distribution and localization

In humans, mRNA expression screening revealed the highest SGLT3 expression in the small intestine, followed by significant expression in skeletal muscle and testis. However, mRNA has also been detected at lower levels in many other tissues, including the adrenal gland, bone marrow, heart, kidney, lung, prostate, spinal cord, stomach, thyroid gland, trachea, uterus, brain and blood vessels (Nishimura & Naito, 2005; Chen et al. 2010). Expression has also been detected in several human colonic and ovarian cancers and in various cancer cell lines (Veyhl et al. 1998).

Human SGLT3 protein has been detected by Western blotting and immunohistochemistry in small intestine, skeletal muscle and kidney (Díez-Sampedro et al. 2003). Immunofluorescence staining localized the protein to discrete patches in the submucosa of the small intestine, which co-localized with the nicotinic acetylcholine receptor, suggesting that hSGLT3 expression is in cholinergic neurones of the submucosal and myenteric plexus. Moreover, similar co-localization was found at the neuromuscular junction of skeletal muscle (Díez-Sampedro et al. 2003). SGLT3 protein has also been reported in human kidney homogenates and the human kidney 2 (HK-2) proximal tubule cell line (Kothinti et al. 2012). Of note, there is a discrepancy between the sizes of the bands detected by these groups. Díez-Sampedro et al. (2003) detected the band at ∼60 kDa, which is smaller than the predicted protein size (72 kDa) that was detected by the other group in the kidney (Kothinti et al. 2012). The antibodies of both groups were made to order and raised against very similar peptides with 95% shared sequence homology. Nevertheless, both groups did provide appropriate controls using overexpressing cell line/oocytes, empty vectors, blocking peptides and negative cross-reactivity with SGLT1.

In the mouse and rat, mRNA expression of SGLT3a and SGLT3b seems to be broadly similar. Using RT–PCR, both isoforms have been found in mouse small intestine and kidney (Gribble et al. 2003; Tabatabai et al. 2003). Quantitative PCR revealed much higher mRNA expression of SGLT3a and SGLT3b in small intestine compared with kidney (∼150,000- and 700-fold, respectively; Barcelona et al. 2012). In the hepatic portal area, which itself is not well defined and includes the portal vein, hepatic artery and common bile duct, the expression levels for both isoforms were similar to levels found in the kidney, with SGLT3a approximately four times higher than SGLT3b (Delaere et al. 2013). The first attempt to profile SGLT3 mRNA expression in rat intestine was reported by Freeman et al. (2006). The authors detected transcripts in duodenum, jejunum and colon, with relatively similar levels detected in each segment. However, the primers and probe sequence for SGLT3 were based on the human sequence for SLC5A4, and although it shares substantial similarities with the rat, it cannot distinguish between the rodent SGLT3a and SGLT3b isoforms. Studies designed to differentiate between the two isoforms have detected expression in hypothalamus, kidney and duodenum in Sprague–Dawley rats (O'Malley et al. 2006), whereas SGLT3b was also detected in jejunum and proximal ileum (Pal et al. 2015). In addition, SGLT3b was detected in different parts of the jejunum after Roux-en-Y gastric bypass (RYGB) surgery in Sprague–Dawley and Zucker diabetic fatty rats (Bhutta et al. 2014). In cell lines, expression was found in the intestinal endocrine GLUTag cell line (Gribble et al. 2003) and in primary cultures of mouse cortical kidney cells (Tabatabai et al. 2001). Overall, from published microarray data sets reporting expression across most tissues, it is clear that the highest expression occurs in the small intestine for both isoforms. Low-level expression is detected in most other tissues, with moderate expression of SGLT3b occurring in testes and ovary (GEO data sets: GDS3142, GDS3357 and GDS4319).

In rodents, there are limited data on SGLT3 at the protein level. It has been localized by Western blot to kidney, liver, ileum and the portal vein area. However, the specificity of the antibody is uncertain, because the authors interpreted two bands of 71 and 72 kDa as staining for both SGLT3a and SGLT3b isoforms, but did not describe how the antibody was generated (Delaere et al. 2013). SGLT3 has also been detected by Western blot in various brain structures, including cortex, hypothalamus, hippocampus, midbrain, striatum, medulla, olfactory bulb and cerebellum (Yamazaki et al. 2014). The antibody used was raised against the peptide for mouse SGLT3b, and again, the authors could not distinguish between the SGLT3a and SGLT3b isoforms. Using immunofluorescence, the authors detected SGLT3 in neurones, but not astrocytes of the striatum and cortex; detection was performed using another SGLT3 antibody, although details of its specificity were not provided. However, the staining strongly co-localized with choline acetyltransferase, which is consistent with the co-localization of hSGLT3 and the β-subunit of the acetylcholine receptor reported in human skeletal muscle and intestine (Díez-Sampedro et al. 2003), suggesting that cholinergic neurones are an important site of mSGLT3 protein localization. Although neuronal localization is likely in these tissues, wider neuronal localization has not been explored.

Taken together, the highest expression among tissues is convincingly reported at the mRNA level in different regions of the small intestine. Ubiquitous expression at low levels seems to be present in most tissues, with possibly more significant expression in the ovary, testis and skeletal muscle. Though expression has been detected in the kidney, its level seems to be very low, and the localization of mRNA expression to specific cell types is lacking. Western blotting has revealed SGLT3 protein in small intestine, kidney, brain structures, liver and the portal vein area. Human SGLT3 protein has been localized to cholinergic neurones of the small intestine submucosal and myenteric nerve plexuses, and neuromuscular junction of skeletal muscle. In the mouse, cholinergic neurones of brain cortex and striatum also show positive immunofluorescence staining. However, localization data at the protein level need to be considered with some caution, because the antibodies used do not always show convincing specificity or distinguish between isoforms, and additional studies on cellular localization are required. The expression of all isoforms is summarized in Table 3.

Table 3. Tissue distribution of expression of different isoforms
Messenger RNA Protein
Isoform RT–PCR Quantitative PCR Western blot Immunohistochemistry
Human SGLT3 Small intestinea, colonb, skeletal musclea, kidney cell linea,c Small intestined,e, kidneyd,e, adiposee, adrenald, blood vessele, boned, braine, heartd,e, lungd, ovarye, prostated, skeletal musclee, spleene, stomachd,e, testisd,e, thyroidd, trachead Small intestinea, kidneyc, kidney cell linec, skeletal musclea Small intestinea, skeletal musclea
Mouse SGLT3a Small intestinef,g, intestinal cell linef,g, kidneyh Small intestinei, kidneyi,j, portal areah Small intestinej, kidneyj, liverj, portal areaj
Mouse SGLT3b Small intestineg, kidneyh, kidney cell linek Small intestinei, kidneyi,j, portal areaj Small intestinej, kidneyj, brainl, liverj, portal areaj Brainl
Rat SGLT3a Small intestinem, kidneyi, brainm Small intestinen, colonn
Rat SGLT3b Small intestinem, brainm Small intestineo,p
  • aDíez-Sampedro et al. (2003); bVeyhl et al. (1998); cKothinti et al. (2012); dNishimura et al. (2005); eChen et al. (2010); fGribble et al. (2003); gLee et al. (2015); hTabatabai et al. (2003); iBarcelona et al. (2012); jDelaere et al. (2013); kTabatabai et al. (2001); lYamazaki et al. (2014); mO'Malley et al. (2006); nFreeman et al. (2006); oBhutta et al. (2014); and pPal et al. (2015).

Functional properties

Functional studies in Xenopus laevis oocytes expressing human SGLT3 showed that hSGLT3 does not transport glucose in the absence or presence of Na+ at pH 7.5 or 5 (Díez-Sampedro et al. 2003); however, d-glucose and α-MDG were able to generate a phlorizin-sensitive, Na+-dependent depolarization of oocyte cell membrane potential, whereas d-galactose, d-fructose and mannitol had no effect. The glucose-induced depolarization shows saturation with a K0.5 of 20 mm and a maximal depolarization of 23 mV, increasing as the resting membrane potential is lowered (Díez-Sampedro et al. 2003). No glucose/α-MDG-induced currents were observed in the absence of Na+ at pH 7.4, although glucose/α-MDG-induced currents are substantially greater at lower pH (pH 5) when the charge carrier is likely to be H+. In addition, at low pH, glucose/α-MDG is able to induce currents even in Na+-free media. This is supported by the observation that there is no increase in Na+ uptake at pH 5, but there is significant intracellular acidification, consistent with H+ as the charge carrier at low pH. Interestingly, the imino sugars, 1-deoxynojirimycin (DNJ), N-hydroxylethyl-1-deoxynojirimycin (miglitol) and N-butyl-1-deoxynojirimycin (miglustat), which are all potent inhibitors of α-glucosidase enzymes of the intestinal brush border membrane, are potent agonists at hSGLT3 with a K0.5 of 0.5–5 μm, which is up to 40,000 times greater affinity than for glucose (Voss et al. 2007). However, imino sugars are not of mammalian origin and are not expected to be the natural agonists. Nonetheless, it provides a pharmacological tool to study a functional role of SGLT3, although their higher affinities for α-glucosidases prevent their use as specific agonists, at least in the small intestine.

In rodents, the two isoforms of SGLT3 exhibit different electrophysiological properties, as well as different responses to glucose and glucose analogue-induced stimulation. At pH 7.4 in the presence of Na+, mSGLT3a expressed in oocytes does not depolarize the cell membrane when exposed to d-glucose, α-MDG or DNJ, unlike hSGLT3. In contrast, at pH 7.4, glucose, α-MDG, 1-deoxyglucose and 6-deoxyglucose induce a strong depolarization in mSGLT3b-expressing oocytes (Aljure & Díez-Sampedro, 2010; Barcelona et al. 2012). Like mSGLT3a, mSGLT3b is also not activated by DNJ, but unlike hSGLT3 and mSGLT3a, mSGLT3b is able to transport sugar to a small extent, although the uptake of the glucose analogue α-MDG is about 60 times less than uptake by mSGLT1 (Aljure & Díez-Sampedro, 2010). In addition, mSGLT3b shows only a small pH dependency; glucose-induced currents are present at neutral and acidic pH (Barcelona et al. 2012). Rat SGLT3a exhibits similar functional properties to mSGLT3a. Oocytes expressing rSGLT3a exposed to glucose showed minimal currents at pH 7.4, but inward currents are much larger at lower pH, and in the presence of glucose (Barcelona et al. 2012). These characteristics of mSGLT3a and mSGLT3b in oocytes have been confirmed in mammalian CHO (Chinese hamster ovary) cells expressing these isoforms (Barcelona et al. 2012). It seems that in rodents, the properties of the SGLT3 isoforms can be separated according to external pH; however, the significance of this in vivo is unknown.

Overall, there are substantial differences between human and rodent SGLT3 electrophysiological characteristics that are summarized in Table 4. Although hSGLT3 does not transport glucose, it can induce a small depolarization at pH 7 and it is pH sensitive. In contrast, mouse and rat SGLT3a do not respond to glucose at pH 7, but at low pH can generate currents carried most probably by H+, and these glucose-induced currents are larger than those for H+ alone. In contrast, mSGLT3b can transport glucose and generates currents in response to glucose, rather than H+.

Table 4. Table of sodium–glucose transporter (SGLT) isoform 3 characteristics and differences
Characteristic hSGLT3 r/mSGLT3a mSGLT3b
Glucose uptake No No Small
pH sensitivity Yes Yes No
Phlorizin sensitivity Yes No Yes
Glucose/α-MDG-induced currents at pH 7.4 Yes, small No Yes, large
Glucose/α-MDG-induced currents at pH 5 Yes, large Yes, large Yes, small
H+-induced currents Yes, medium Yes, large No
Na+ requirement Yes, partial No Yes, partial
DNJ-induced currents at pH 7.4 Yes No No
DNJ-induced currents at pH 5 Yes, large Yes n.d.
  • Abbreviations: α-MDG, α-methyl-d-glucose; DNJ, 1-deoxynojirimycin; and n.d., not determined, data are not available. Sources: Díez-Sampedro et al. (2003); Voss et al. (2007); Aljure & Díez-Sampedro (2010); Barcelona et al. (2012).

Physiological role

Although the electrophysiological properties of human and mouse SGLT3 have been well described, their physiological function is unknown. The inability of SGLT3 protein to transport glucose (or its analogues) and its ability to generate membrane currents in the presence of glucose and Na+ or H+ have led to the hypothesis that SGLT3 might act as a glucose sensor. Another hypothesis is that SGLT3 controls gastric emptying, which may be related to the pH dependency of membrane current generation by SGLT3 (see earlier); chyme released from the stomach will progressively lower duodenal luminal pH (Miller et al. 1978) causing SGLT3 to generate larger currents at this lower pH. Intraduodenal perfusion of glucose and its analogues 3-O-methylglucose (3-OMG) and α-MDG can inhibit gastric emptying in conscious rats, whereas 2-deoxyglucose (a substrate for facilitative glucose transporters) cannot (Freeman et al. 2006). In addition, intraduodenal perfusion with glucose inhibits gastric motility in anesthetized rats, whereas galactose has the opposite effect. The authors (Freeman et al. 2006) used the difference in glucose and galactose affinity for SGLT1 and SGLT3 (both glucose and galactose are substrates of SGLT1, whereas galactose has negligible affinity for SGLT3; Díez-Sampedro et al. 2000; Voss et al. 2007) to conclude that SGLT3 delays gastric emptying. However, the low affinity of galactose for hSGLT3 was seen at pH 7.4, whereas the ability of galactose to induce currents via mSGLT3a at pH 5 is similar to that of glucose and other glucose analogues (Barcelona et al. 2012). Although the same authors consider 3-OMG and α-MDG to be substrates for both SGLT1 and SGLT3, others have reported data showing 3-OMG is not a substrate for pig SGLT3 (Díez-Sampedro et al. 2000) and interpret the difference between 3-OMG and α-MDG to distinguish the effects between SGLT1 and SGLT3 in rats (Delaere et al. 2013; Pal et al. 2015). Thus, the interpretation of SGLT3 as a mediator of gastric emptying by Freeman et al. (2006) seems less certain.

The role of SGLT3 as a glucose sensor has also been proposed and tested in the hepatic portal vein of the rat (Delaere et al. 2013). Detection of glucose in the hepatic portal vein after protein-enriched, diet-induced intestinal gluconeogenesis has been shown to decrease food intake (Mithieux et al. 2005). Delaere and colleagues (2013) excluded GLUT2 and taste receptors as mediators of the sensing mechanism and proposed SGLT3 as the sensing mediator. They showed that glucose or α-MDG infusion reduced food intake, whereas 3-OMG did not. The effect of glucose on food intake was abolished by phlorizin and following exposure of the portal vein to capsaicin to denervate sensory nerves. The effect of glucose was not changed after ventral vagotomy. SGLT3 mRNA and SGLT3 protein were detectable in the portal vein area; however, the reported mRNA levels were similar to those found in the kidney, which are much lower than in intestine (Barcelona et al. 2012; Soták M., unpublished observations). As the presence of SGLT3 can be detected, and sensory afferents are involved, and because 3-OMG is not a substrate for SGLT3, at least in the pig, these authors concluded that SGLT3 is the portal glucose sensor.

The main glucose-sensing mechanism in the body that leads to insulin secretion depends on closure of ATP-sensitive K+ channels in pancreatic β-cells. A similar mechanism has been proposed for glucose stimulation of neurones. However, only a small proportion (9%) of cultured hypothalamic glucose-stimulated neurones show any response to the K+ channel inhibitor tolbutamide (O'Malley et al. 2006), suggesting that other mechanisms exist. The majority of glucose-excitable cultured hypothalamic neurones show a rise in cytosolic Ca2+ in response to the α-MDG, a substrate of the SGLTs, but not the GLUTs; an effect blocked by phlorizin (O'Malley et al. 2006). A higher proportion of neurones are activated by α-MDG (67%) than by 3-OMG (45%). This difference may be accounted for by SGLT3 and is supported by the finding of SGLT3a and SGLT3b expression in rat hypothalamus and cultured hypothalamic neurones (O'Malley et al. 2006). Moreover, hSGLT3 expressed in sensory neurones of Caenorhabditis elegans is able to mediate preferential glucose-induced chemotaxis at low pH, and this effect is blocked by phlorizin (Bianchi & Díez-Sampedro, 2010). In addition, glucose and DNJ, an hSGLT3 agonist, activate rat intestinal enterochromaffin cells and myenteric neurones (Vincent et al. 2011).

Roux-en-Y gastric bypass surgery

Roux-en-Y gastric bypass surgery is a highly effective treatment for severe obesity, especially in diabetic patients. Isolation of the proximal small intestine and its luminal glucose-sensing capacity from nutrients, combined with the presence of non-digested luminal nutrients in more distal parts of the small intestine, is thought to be crucial in the weight-reducing and anti-diabetic effects of surgery, although the mechanisms have not been defined. SGLT3b mRNA is expressed differentially along the small intestine after RYGB surgery in a rat model (Bhutta et al. 2014). Expression was significantly higher in the common limb (distal to the Y-intersection) of both Sprague–Dawley and diabetic Zucker diabetic fatty rats compared with the biliopancreatic (bypassed portion of stomach and duodenum) and Roux limbs (anastomosis of fundal gastric remnant with jejunum). In contrast, no differential mRNA expression was detected for SGLT1, GLUT2 and taste receptor T1R2. Furthermore, infusion of saccharin, which stimulates taste receptors T1R2/3, into the biliopancreatic limb downregulated expression of SGLT3b in the common limb of Zucker diabetic fatty rats, though not significantly in Sprague–Dawley rats (Bhutta et al. 2014). These data suggest a possible role for SGLT3 in the post-RYGB intestinal changes.

The involvement of SGLT3-mediated intraluminal glucose sensing in proximal small intestine has also been proposed in a foregut exclusion model resembling RYGB (Pal et al. 2015). Here, the proximal duodenojejunal part of the intestine was isolated so that different glucose analogues could be administered to the excluded segment. At the same time, a glucose bolus was administered to the jejunum so that the effect on absorption could be measured. Administration of α-MDG caused significantly higher glucose absorption than 3-OMG or saccharin, and this effect was blocked by the SGLT1/SGLT3 inhibitor phlorizin. The authors conclude that SGLT3, and not SGLT1 or taste receptors, mediates the higher glucose absorption in the RYGB model. Overall, it seems that SGLT3 may have a role in the beneficial outcomes of RYGB, although more direct evidence is required.

Glucagon-like peptide-1 secretion

SGLT3 has been thought to play a role in the secretion of the incretin hormone glucagon-like peptide-1 (GLP-1). In the foregut exclusion model, GLP-1 secretion (measured as the difference between portal and systemic concentration) was significantly higher after treatment with the SGLT1/3 agonist α-MDG, whereas no difference was detected after treatment with the SGLT1-only agonist 3-OMG (Pal et al. 2015), suggesting SGLT3 might mediate GLP-1 secretion. The effect of α-MDG was blocked by phlorizin and prior vagotomy, supporting SGLT participation and indicating involvement of vagal innervation.

An indirect role for SGLT3 in mediating GLP-1 secretion was proposed recently by Lee et al. (2015). To stimulate SGLT3, the authors used miglitol, an iminosugar that is used as an α-glucosidase inhibitor and is a potent agonist of human SGLT3 (Voss et al. 2007; Lee et al. 2015). Miglitol was able to activate duodenal enteroendocrine cells more effectively than acarbose, another α-glucosidase inhibitor. However, miglitol administration alone failed to increase GLP-1 secretion enough to alter plasma concentrations and was effective only when co-administered with maltose. Since no GLP-1 stimulation was seen after giving acarbose with maltose, it was concluded that miglitol may have a direct effect on SGLT3 to stimulate GLP-1 secretion (Lee et al. 2015).

Some controversies and limitations

Given that there is still a lack of suitable tool compounds for studying SGLT3 function directly, so far all reports on its likely function in vivo are based on indirect evidence. Several authors (O'Malley et al. 2006; Delaere et al. 2013; Pal et al. 2015) argue for discrimination between SGLT1 and SGLT3 on the basis of differing affinities for α-MDG and 3-OMG; α-MDG is a substrate for both SGLT1 and SGLT3, whereas 3-OMG is a substrate for SGLT1 only. However, this selectivity is based only on data from pig SGLT3 (Díez-Sampedro et al. 2003), and we do not know if it extends to other species. Likewise, the putative SGLT3 agonist miglitol was shown to activate human SGLT3 (Voss et al. 2007; Lee et al. 2015), although evidence for this in rodents is lacking, despite its use in rats (Lee et al. 2015). Moreover, some authors have not tried or not been able to distinguish between the SGLT3a and SGLT3b rodent isoforms in their studies, particularly when using antibodies for which the isoform specificity is unclear, or they have focused on only one isoform (SGLT3b).

Future perspectives

A glucose-sensing mechanism for SGLT3 has been proposed from in vitro models and based on the ability of SGLT3 to generate membrane currents in the presence of glucose, Na+ and/or H+. However, direct evidence for glucose sensing in vivo is still lacking. Neuronal protein expression has been shown in human submucosal/myenteric tissue, and mRNA expression has been detected in scraped mucosa, and by microarray gene expression analysis in enterocytes and enteroendocrine cells. Tissue localization of rodent isoforms remains controversial at the protein level, because of antibody specificity, and poorly defined mRNA expression. A more definitive examination of the organ and tissue distribution of SGLT3 is still needed, as well as better selective agonists and antagonists, or its function will remain intriguing, but increasingly speculative. Knockout mice for both rodent isoforms have been reported from one source to have no obvious phenotype, but they have not been investigated in any detail and are not widely available for study. Emerging technologies, such as CRISPR, make it possible to produce isoform-specific knockout mice or even rats, which in combination with imino sugars and specific SGLT inhibitors, should enable us to explore and define better the physiological and pathophysiological function of SGLT3, at least in rodents.

Biography

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    Matúš Soták is a postdoctoral fellow at AstraZeneca, where he pursues explorative research of novel isoforms of sodium–glucose transporters and their possible roles in metabolic pathophysiology. He received a PhD at the Faculty of Science, Charles University in Prague, Czech Republic. As a PhD student and research student at the Institute of Physiology, Czech Academy of Sciences, he investigated the role of the circadian clock in regulation of intestinal function, and the results were published in a few respectable journals. His contributions were awarded a young investigator prize at an international conference and the best publication of the year at his former institute.

Additional information

Competing interests

None declared.

Author contributions

M.S. and R.J.U. designed, conceived and drafted the manuscript. M.S. performed sequence alignment analyses. M.S., J.M. and R.J.U. revised the manuscript critically for important intellectual content, approved the final version of the manucript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

Funding

M.S. and R.J.U. are employees of AstraZeneca.

Acknowledgements

We thank Nick Oakes (AstraZeneca, Mölndal) for valuable discussions and comments and the reviewers for their insightful suggestions.