Aouameur R, Da Cal S, Bissonnette P, Coady MJ, Lapointe J-Y. SMIT2 mediates all myo-inositol uptake in apical membranes of rat small intestine. Am J Physiol Gastrointest Liver Physiol 293: G1300-G1307, 2007. First published October 11, 2007; doi:10.1152/ajpgi.00422.2007.-This study presents the characterization of myo-inositol (MI) uptake in rat intestine as evaluated by use of purified membrane preparations. Three secondary active MI cotransporters have been identified; two are Na ϩ coupled (SMIT1 and SMIT2) and one is H ϩ coupled (HMIT). Through inhibition studies using selective substrates such as D-chiro-inositol (DCI, specific for SMIT2) and L-fucose (specific for SMIT1), we show that SMIT2 is exclusively responsible for apical MI transport in rat intestine; rabbit intestine appears to lack apical transport of MI. Other sugar transport systems known to be present in apical membranes, such as SGLT1 or GLUT5, lacked any significant contribution to MI uptake. Functional analysis of rat SMIT2 activity, via electrophysiological studies in Xenopus oocytes, demonstrated similarities to the activities of SMIT2 from other species (rabbit and human) displaying high affinities for MI (0.150 Ϯ 0.040 mM), DCI (0.31 Ϯ 0.06 mM), and phlorizin (Pz; 0.016 Ϯ 0.007 mM); low affinity for glucose (36 Ϯ 7 mM); and no affinity for L-fucose. Although these functional characteristics essentially confirmed those found in rat intestinal apical membranes, a unique discrepancy was seen between the two systems studied in that the affinity constant for glucose was ϳ40-fold lower in vesicles (Ki ϭ 0.94 Ϯ 0.35 mM) than in oocytes. Finally, the transport system responsible for the basolateral efflux transporter of glucose in intestine, GLUT2, did not mediate any significant radiolabeled MI uptake in oocytes, indicating that this transport system does not participate in the basolateral exit of MI from small intestine. brush border; glucose; transport; oocytes; phlorizin THE PHYSIOLOGICAL IMPORTANCE of myo-inositol (MI) is generally considered to reflect its role in signal transduction as a precursor to phosphoinositides and inositol phosphates. In addition, MI acts as a "compatible osmolyte" in specific tissues, such as brain and kidney medulla, where variations in milieu osmolarity may threaten normal cell function. In response to an increase in osmolarity, intracellular MI concentration may rise up to 500-fold above its plasma concentration of ϳ30 M (9, 11, 12), where it prevents the accumulation effects of high ionic concentrations, which leads to DNA degradation (11a). This has been well documented in brain where conditions such as trauma (30), edema and hypernatremia (26,27,38) have been shown to increase MI levels. To reach such high intracellular concentrations, secondary active transport systems are required for MI.

Myo-inositol (MI) is involved in several important aspects of cell physiology including cell signaling and the control of intracellular osmolarity i.e. by serving as a "compatible osmolyte". Currently, three MI cotransporters have been identified: two are Na(+)-dependent (SMIT1 and SMIT2) and one is H(+)-dependent (HMIT) and predominantly expressed in the brain. The goal of this study was to characterize the expression of SMIT2 in rabbit kidney and to compare it to SMIT1. First, we quantified mRNA levels for both transporters using quantitative real-time PCR and found that SMIT1 was predominantly expressed in the medulla while SMIT2 was mainly in the cortex. This distribution of SMIT2 was confirmed on Western blots where an antibody raised against a SMIT2 epitope specifically detected a 75 kDa protein in both tissues. Characterization of MI transport in brush-border membrane vesicles (BBMV), in the presence of d-chiro-inositol and l-fucose to separately identify SMIT1 and SMIT2 activities, showed that only SMIT2 is expressed at the luminal side of proximal convoluted tubules. We thus conclude that, in the rabbit kidney, SMIT2 is predominantly expressed in the cortex where it is probably responsible for the apical transport of MI into the proximal tubule.

This study presents the characterization of MI uptake in rat intestine using purified membrane preparations (BBMv). The two Na+‐coupled myo‐inositol (MI) cotransporters identified (SMIT1 and SMIT2) can be differentiated through inhibition studies using the selective substrates D‐chiro‐inositol (DCI, specific for SMIT2) and L‐fucose (specific for SMIT1). Results show that SMIT2 is exclusively responsible for apical MI transport in rat intestine. Other sugar transport systems present in apical membranes (SGLT1 and GLUT5) lacked any significant contribution to MI uptake. Functional analysis of rat SMIT2 activity determined using electrophysiological studies in Xenopus oocytes, demonstrated similarities to the activities of SMIT2 from species (rabbit and human), displaying high affinities for MI (0.150 ± 0.040 mM), DCI (0.31 ± 0.06 mM) and Pz (0.016 ± 0.007 mM), low affinity for glucose (36 ± 7 mM) and no affinity for L‐fucose. Electrophysiological studies essentially confirmed those found in rat intestinal BBMv with exception of glucose affinity which was about 40‐fold higher in vesicles (Ki = 0.94 ± 0.35 mM) when compared to oocytes. Finally, GLUT2 expressed in oocytes did not mediate any significant radiolabelled MI uptake, indicating that this transport system does not participate in the basolateral exit of MI from small intestine.

Myo‐inositol (MI) uptakes in cells is essentially performed by two Na+‐coupled transport systems; SMIT1, a basolateral system inducible through hypertonicity and SMIT2, an apical system responsible for MI uptake in the kidney and intestine, amongst other tissues. In diabetes, urinary MI excretion is increased by 1 order of magnitude through a non‐established mechanism. The purpose of this study is thus to investigate the influence of an acute diabetic condition (5‐day streptozotocintreatment) on the expression of both SMIT mRNA's in rat brain, liver, muscle, kidney and intestine through quantitative‐RT‐PCR (qRT‐PCR). Also, SMIT2 activity was determined by measuring MI transport in purified brush border membranes (BBMv) from kidney and intestine. SMIT1 transcripts are mainly found in the kidney where it doubles in diabetic rats. Other tissues show more modest mRNA levels and were not altered in diabetes. SMIT2 transcripts were found in all tested tissues in varying levels but were not modified in diabetes. MI uptakes performed on kidney and intestine BBMv confirmed the qRT‐PCR results showing no variations of transport in STZ rats due to SMIT2. We thus conclude that the increase in MI urinary excretion is not due to a reduction in the apical expression of MI cotransporter SMIT2.