Generation of the sodium/myo-inositol cotransporter 1 (Slc5a3) gene murine knockout model, characterization of its lethal phenotype, and revision of the 'inositol depletion hypothesis'.

摘要

Myo-inositol (Ins) is a ubiquitous hexahydroxycyclohexane in living organisms, and the most abundant in mammalian cells of all nine possible stereoisomers along with scyllo-, neo-, epi-, muco-, allo-, cis-, and the enantiomers, D-chiro and L-chiro. Ins is an important osmolyte and a key component of phosphatidylinositol (PtdIns), unique in the family of phospholipids in that its Ins headgroup can be further phosphorylated to generate various phosphoinositides. Among them, phosphatidylinositol-4,5-bisphosphate (Ptdlns-4,5-P2) is involved in important signal transduction events. Ins's cellular uptake is carried out by a system of specific membrane transporters, of which the SLC5A3 (SMITI), a Na+/myo-inositol cotransporter, is the one with the highest affinity for the polyol.;The principle aim of my thesis work is to test the widely accepted " myo-inositol depletion hypothesis" envisioned to explain the mechanism of action of lithium, complications of diabetes and hereditary galactosemia. This assumption, which states that a reduction of intracellular myo -inositol results in a decreased PtdIns synthesis, and therefore a disruption of cellular signaling, had never been tested by direct measurements of PtdIns. To investigate the effect of Ins deficiency at the cellular level, a murine knockout model of the Slc5a3 gene encoding the Na+/Ins cotransporter was generated and studied. The resulting Slc5a3-null mice die shortly after birth, although the phenotype can be rescued by supplementation of 1% Ins in the drinking water of the pregnant dam. The rescued Slc5a3 knockout mice survive only if the Ins enriched water treatment continues until weaning, 14 days after birth.;My work shows that a 69.7% reduction in myo-inositol is responsible for a 47% reduction of PtdIns in the murine Slc5a3--null fetus at embryonic stage 10.5, but the hypothesis proves to be invalid at later stages of development, at embryonic stages 14.5 and at 18.5, and through adulthood. The work outlined in this dissertation also shows that the 'Ins depletion hypothesis', as it has been proposed, proves to be invalid since Ins levels in mammalian tissues have never been shown to be reduces to the degree observed in Slc5a3 knockout mouse model used in my study.