All tissues used in this study were obtained from ethically approved intermediaries. Tissue supply was governed by legal agreements and by stringent ethical review from local research ethics committees. In addition, and in all cases, the informed consent of the donor or the donor’s next of kin was obtained for the use of the donated tissue for research. In all, 46% of the tissues were from female donors while 54% were from male donors. The age distributions of the tissue donors were: <20 years old: 5%; 20–39 years old: 17%; 40–60 years old: 32%; 60–80 years old: 31%; >80 years old: 14%. No ethnicity information relating to tissue donors was available.
The genes that were profiled by quantitative RT-PCR for their expression levels across a panel of 72 different human tissues are listed by both their common name and their systematic name in Table 1A. Also listed are the putative substrates for each of the transporters, as well as the chromosomal location of each transporter gene. The primer sequences used for PCR were designed to be general for all known isoforms and splice variants of each gene. The sequences of the amplicons, the exon location of the amplicons, and their precise genomic coordinates are listed in Table 2.31 Included in each PCR reaction was a primer/probe set for the GAPDH gene. This was done to control for the success of the first strand cDNA synthesis reaction and the eventual PCR. These data were not used for normalization purposes since GAPDH levels themselves vary considerably across the tissue type and individual donor.30 The study included total RNA isolated from three different individuals for each tissue in a panel of 72 tissues. The 72 tissues in the study extend across all major human biological systems: cardiovascular, digestive, endocrine, male and female reproductive, hematopoietic and lymphatic, integumentary, musculoskeletal, nervous, respiratory, and urinary systems.30 The individual RNA samples used in the study were prepared from normal tissues, although their donors may have had abnormal or diseased symptoms in other tissues or organs. The donors represent different genders and age groups, and every attempt was made to use the same samples for the analysis of all seven SGLT family members. A few exceptions did occur which bear no impact on our study conclusions.
Tissue expression profiling of the seven SGLT family members reveals distinct patterns of tissue expression, summarized below.
The Kidney-Specific and Kidney-Abundant SGLT Family Members
Figure 1A presents the results evaluating the expression of SGLT2 from a subset of the 72 tissues profiled (data from all the tissues are available in the Appendix, Additional Figure S1) with a primer/probe set (SGLT2-e6,7) designed to span the boundaries of exons 6 and 7 of SGLT2, as noted in Table 2). In all figures presented, the Y-axis represents the number of transcripts per μg of RNA. The tissue with the highest level of expression was the kidney cortex where the expression was approximately 300-fold higher than the tissue with the next highest level of expression, the kidney medulla. Although small numbers of putative transcripts could be observed in some of the other tissues, no evidence of SGLT2 expression was observed in the majority of other tissues, including the 20 brain subregion RNA samples tested (eight of these are shown in Figure 1A). These data are consistent with several other reports evaluating the pattern of expression of SGLT2 in human tissues.9,10,16 The GAPDH data, plotted in log2 format on the right-hand y-axis of the panel of Figure 1A, indicates that all tissue RNA samples had successful first strand synthesis and PCR reactions. The error bar associated with the tissue data indicates the variation observed in expression measurements using individual RNA samples from three individual donors and not that obtained with technical replicates. Our study methodology is thus different from other studies reported in the literature where pooled samples from commercial vendors were utilized.22
Similar data was obtained using three additional primer/probe combinations spanning different exon regions (exons 1 and 2, exons 4 and 5, exons 9 and 10) of the SGLT2 gene (see the Appendix, Additional Figures S2–S4), further supporting that the expression of the SGLT2 gene is highly specific for the kidney cortex. Low transcript levels were detected across a range of tissues outside the kidney for the primer/probe set SGLT2-e4,5 (Table S2), possibly due to greater primer/probe set efficiency; however, even in this case, kidney expression was 100-fold higher than in the next highest tissue observed (the ileum). In addition, the primer/probe combination (SGLT2-e13), located in exon 13 close to the 3’ end of the SGLT2 gene and identical to the one used by Zhou et al.,22 shows that the SGLT2 gene is highly specifically expressed in the kidney (Figure 1B, Figure S5, and Table S2), unlike the result previously reported by these investigators. We observed that this primer/probe set essentially mirrors the data obtained with the primer/probe set spanning the junction of exons 6 and 7, as well as primer/probe sets spanning the exons 1–2, 4–5, and 9–10. We note that the primer/probe SGLT2-e13 lies in a region of overlap with another SGLT2-unrelated transcript, and thus is less specific to the SGLT2 sequence compared with other SGLT2 primer/probes we tested. The potential consequences of using this probe are further discussed in “the SGLT2 locus” section below. In summary, these data suggest that the steady state levels of SGLT2 transcripts are highly specific for the cortex of the kidney in humans.
SGLT5, a relatively uncharacterized SGLT family member, is also found to have a highly kidney-abundant tissue expression pattern. The expression of SGLT5 in a subset of the 72 human tissues is shown in Figure 2A (data from all 72 human tissues can be found in the Appendix, Additional Figure S6). The highest expression level of SGLT5 is found in the kidney cortex, while in the kidney medulla SGLT5 is expressed at about half of the level found in the kidney cortex. Because the extremely high level of kidney expression may obscure the magnitude of expression observed in other tissues, we replotted the data without the kidney cortex and medulla data in Figure 2B. Unlike the profile observed for SGLT2, which has little or no detectable level in tissues other than the kidney cortex and medulla, SGLT5 exhibits a low level of expression in some tissues like the kidney pelvis, vas deferens, left atrium of the heart, skin, and testes. However, the expression of SGLT5 in the kidney cortex is still 35 times higher than that observed in the vas deferens. These data suggest that the expression of SGLT5 in human tissues is highly abundant in kidney, compared with other tissues.
The Small Intestine and Muscle-Abundant SGLT Family Members
As shown in Figure 3A, the expression of SGLT1 (SLC5A1), the closest homolog to SGLT2, is essentially restricted to the small intestine, the skeletal muscle, and the heart (the data for all 72 human tissues are in the Appendix, Additional Figure S7 and Additional Table S2). Minor numbers of transcripts are observed in the trachea, prostate, cervix, and mesenteric adipose tissue, but like SGLT2, we see no evidence for any expression in the brain subregions tested. The GAPDH data indicates successful first strand synthesis and PCR in all samples. The expression pattern of SGLT1 across human tissues we observed above is generally consistent with reports from other studies using mRNA-based methods.6,7,9 It is worth noting that brain expression of SGLT1 has been reported in other species (rats and pigs) using immunological, in situ hybridization and RT-PCR techniques.29,32,33
Interestingly, the glucose sensor SAAT1 (SLC5A4) and the low-affinity glucose/mannose cotransporter SGLT4 (SLC5A9) display a similar high level of expression in the small intestine (duodenum, jejunum, and ileum) as well as in skeletal muscle (Figure 3B; data for all 72 human tissues are in the Appendix, Additional Figures S8 and S9). In the case of SAAT1, the highest expression could be found in the jejunum, and the steady state SAAT1 RNA level there is about 3.5-fold higher than in skeletal muscle. These data are consistent with other reports of SAAT1 expression in the intestine and skeletal muscle.11,13 The steady state SGLT4 RNA level in the ileum is on the order of 5-fold higher than the next highest-expressing tissue outside of the gastrointestinal (GI) tract, the skeletal muscle. The expression of SGLT1, SAAT1, and SGLT4 in the GI tract seems to be enriched in the small intestinal region: duodenum, jejunum, and ileum; whereas these genes are expressed at a much lower level in the large intestine (cecum, colon, and rectum) as well as in other parts of the GI tract such as stomach and oesophagus (Figure 3). Unlike SGLT1, SAAT1 and SGLT4 display a much lower level of expression in the heart. On the other hand, SGLT4 has a uniquely moderate expression level in pancreas compared with SGLT1 and SAAT1. In all other tissues tested, SGLT1, SAAT1, and SGLT4 have a generally low level of expression (see the Appendix, Additional Figures S7, S8, and S9). Overall, the profile observed here for SGLT4 expression across human tissues is similar to that previously reported.16
The Brain Expresses SGLT6
The solute carrier family 5A11 or SGLT6, a cotransporter with substrate specificity for myo-inositol and glucose, seems to be the only SGLT family member, aside from the ubiquitously-expressed SMIT (see next section), that has extensive expression in all the brain subregions tested. We observed high steady state RNA levels of SGLT6 collectively in the brain, with the highest subregion being the substantia nigra where it was found to be expressed at levels 2–5-fold higher than the other regions tested (Figure 4; data for all 72 human tissues are in the Appendix, Additional Figure S10). SGLT6 is also highly expressed in the spinal cord, at a level similar to that in substantia nigra. However, it is not detected in the dorsal root ganglion (DRG). Other tissue RNA samples with notable expression levels of SGLT6 are the small intestine (ileum and jejunum), kidney (cortex and medulla), as well as skeletal muscle. The observed pattern of SGLT6 in these studies appears to be unique among the SGLT family members tested, and if this pattern is similar in rodents, SGLT6 activity could account, at least in part, for the observation of functional SGLT expression in rat brain.29 It is interesting to note that the human genomic location of SGLT6 coincides with a locus associated with the nervous system disorders of infantile convulsions and choreoathetosis, though no disease-associated mutations were found in the exon or intron/exon boundary sequences of the SGLT6 gene.18 The pattern of expression in human tissues described here differs from that initially described by northern blot,18 although brain expression was detected in both studies.
The Ubiquitously Expressed SMIT
The sodium myo-inositol cotransporter SMIT (SLC5A3) shows a ubiquitous pattern of expression with the highest expression level in the medulla of the kidney and the blood vessel of the choroid plexus (Figure 5; data for all 72 human tissues are in the Appendix, Additional Figure S11). The thyroid gland, pineal gland, dorsal root ganglion, and the testes also have high levels of SMIT expression. Overall, the pattern of expression of SMIT is consistent with what has been previously reported,14 and its expression in all tissues examined highlights its potentially important role in the maintenance of osmotic balance within cells.
The SGLT2 Locus
The discrepancy between our results and that reported by Zhou et al.22 prompted us to examine the SGLT2 locus in more detail. The SGLT2 gene resides on chromosome 16 where 14 exons span approximately 7 kilobases of genomic DNA. The last two exons, exon 13 and 14, overlap with exon 13 of a gene that is encoded on the opposite strand, called C16orf58 (Figure 6A). This gene, conserved in plants, invertebrates, and vertebrates, contains 13 exons spanning 20 kb of genomic DNA, and encodes a protein homologous to the Arabidopsis RUS1 gene.34,35 Numerous sequence submissions to Genbank suggest C16orf58 is indeed expressed. Our internal cDNA cloning effort has obtained full-length cDNA clone of C16orf58. The C16orf58 cDNA clone has a long 3’ untranslated region that contains the reverse-complement sequence of exon 13 and 14 of the SGLT2 gene (data not shown). Electronic northern blots using the region of overlap as a ‘probe’ indicates that expressed sequence tags (ESTs) for C16orf58 versus that of SGLT2 can be found in the NBCI database at the relative abundance of 10 transcripts to one (data not shown). We designed a primer/probe set located in exon 8 of C16orf58 and one that is specific for C16orf58 (Figure 6B). Expression profiling analysis with this primer/probe set indicates that this gene is expressed ubiquitously with the highest steady state RNA levels also located in the cortex of the kidney, the cerebellum (Figure 6C), and the thyroid (see the Appendix, Additional Figure S12). The primer/probe set (SGLT2-e13) used by Zhou et al.22 resides in exon 13 of SGLT2, in the area of overlap between SGLT2 and C16orf58 (Figure 6B), suggesting that measurement of both transcripts would be confounding in experiments where poly d(T) was used to prime first strand synthesis in a one-step PCR process. We have used each of the primers employed by these investigators in separate first strand reactions and have shown that the reverse primer is capable of synthesizing kidney cortex abundant SGLT2 cDNA (Figure 1B), however we have been unable to recapitulate the expression pattern seen with the C16orf58-specific primer (Figure 6C) using the Zhou et al.22 forward primer (data not shown).