Expression profiling and immunolocalization of Na+-d-glucose-cotransporter 1 in mice employing knockout mice as specificity control indicate novel locations and differences between mice and rats

Abstract

The expression and localization of sodium-d-glucose cotransporter SGLT1 (SLC5A1), which is involved in small intestinal glucose absorption and renal glucose reabsorption, is of high biomedical relevance because SGLT1 inhibitors are currently tested for antidiabetic therapy. In human and rat organs, detailed expression profiling of SGLT1/Sglt1 mRNA and immunolocalization of the transporter protein has been performed. Using polyspecific antibodies and preabsorption with antigenic peptide as specificity control, in several organs, different immunolocalizations of SGLT1/Sglt1 between human and rat were obtained. Because the preabsorption control does not exclude cross-reactivity with similar epitopes, some localizations remained ambiguous. In the present study, we performed an immunocytochemical localization of Sglt1 in various organs of mice. Specificities of the immunoreactions were evaluated using antibody preabsorption with the Sglt1 peptide and the respective organs of Sglt1 knockout mice. Because staining in some locations was abolished after antibody preabsorption but remained in the knockout mice, missing staining in knockout mice was used as specificity criterion. The immunolocalization in mouse was identical or similar to rat in many organs, including small intestine, liver, and kidney. However, the male-dominant renal Sglt1 protein expression in mice differed from the female-dominant expression in rats, and localization in lung, heart, and brain observed in rats was not detected in mice. In mice, several novel locations of Sglt1, e.g., in eyes, tongue epithelial cells, pancreatic ducts, prostate, and periurethral glands were detected. Using end-point and quantitative RT-PCR in various organs, different Sglt1 expression in mice and rats was confirmed.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-017-2056-1) contains supplementary material, which is available to authorized users.

RS1 (Rsc1A1) deficiency limits cerebral SGLT1 expression and delays brain damage after experimental traumatic brain injury

“…In the MCAO studies changes of SGLT1 protein were analyzed in Western blots using an antibody supplied by Millipore (Yamazaki et al 2012(Yamazaki et al , 2015. However, in our hands the commercial antibodies supplied by Merck Millipore (07-1417, Lot #2730935), Abcam (ab14686), and Santa Cruz Biotechnology (M-19, sc-20582) and also our own custom made antibody against mouse SGLT1 (Gorboulev et al 2012) did not show specific immunocytochemical reaction with the low expressed SGLT1 protein in brain using the SGLT1 À/À mice as controls (Madunic et al 2017). Actually, some immunocytochemical reactions observed in brain Fig.…”

“…) did not show specific immunocytochemical reaction with the low expressed SGLT1 protein in brain using the SGLT1 −/− mice as controls (Madunic et al . ). Actually, some immunocytochemical reactions observed in brain turned out to represent cross‐reactivity because they were removed after preabsorption of the antibody with the antigenic peptides but remained in SGLT1 −/− mice.…”

“…Unfortunately, the immunocytochemical localizations of SGLT1 in brain must be considered with caution because using Sglt1 −/− mice as specificity control for the immunolocalization of SGLT1 in mouse brain indicate that the so far employed antibodies do not allow a specific identification of SGLT1 (Madunic et al . ). A pathophysiological role of (a) SGLT transporter(s) during energy deficiency was demonstrated by showing in a rat model that an uptake of the SGLT‐specific glucose analog methyl‐α‐D‐glucopyranoside was increased in an epileptic focus (Poppe et al .…”

“…Immunocytochemical localization and in situ-hybridization experiments strongly suggest that SGLT1 is expressed in neurons and provide hints on additional locations of SGLT1 in glial cells and the BBB (Poppe et al 1997;Elfeber et al 2004;Vemula et al 2009;Yu et al 2010;Yamazaki et al 2015). Unfortunately, the immunocytochemical localizations of SGLT1 in brain must be considered with caution because using Sglt1 À/À mice as specificity control for the immunolocalization of SGLT1 in mouse brain indicate that the so far employed antibodies do not allow a specific identification of SGLT1 (Madunic et al 2017). A pathophysiological role of (a) SGLT transporter(s) during energy deficiency was demonstrated by showing in a rat model that an uptake of the SGLT-specific glucose analog methyl-a-D-glucopyranoside was increased in an epileptic focus (Poppe et al 1997) and that the lesion volume after middle cerebral artery occlusion (MCAO) mimicking stroke was decreased in the presence of SGLT-specific inhibitor phlorizin (Vemula et al 2009;Yamazaki et al 2012).…”

Sodium-glucose cotransporters: new targets of cancer therapy?

“…G -glucose; CG -cortical glomerulus; CPT -convoluted proximal tubules; JMG -juxtamedullary glomerulus; PT -proximal tubule. The detailed immunohistochemical localization of SGLT2 and SGLT1 in the human kidney was described in our previous publication (23) 23, 24,43). This difference in the molecular weights of SGLT1 could be explained by cell line/tissue-specific pattern or protein glycosylation and/or phosphorylation.…”