br The glycine transporter GlyT was originally identified as

The glycine transporter 1 (GlyT1) was originally identified as a member of the solute carrier family 6 of sodium- and chloride-dependent neurotransmitter transporters . GlyT1 is expressed in the central nervous system and in peripheral tissues; mainly in erythroid cells, from erythroblasts in the bone marrow up to circulating reticulocytes in humans and rats . Reduced glycine transfer may affect hemoglobin (Hb) biosynthesis because glycine is a key rate-limiting component in the first catalytic reaction of the heme synthesis pathway. Heme biosynthesis begins and ends in the mitochondria, involves eight enzymatic reactions, and is highly conserved in mammals . It is initiated with a condensation reaction between glycine and succinyl coenzyme A to form 5-aminolevulinic ALW-II-41-27 sale (ALA). ALA is catalyzed by δ–aminolevulinic acid synthase 2 (ALAS2) in erythroid cells. ALAS2 expression is controlled by erythroid-specific transcription factors that adjust the rate of heme synthesis based on intracellular iron availability . In the final reaction, heme is formed by ferrochelatase catalyzing the insertion of a ferrous iron into a protoporphyrin IX (PPIX) ring. Heme is then exported out of the mitochondria to associate with globin chains and apocytochromes, a process mediated by adenosine triphosphate (ATP)-binding cassette transporters, forming Hb . In vitro studies suggest that GlyT1 is no longer expressed in mature red blood cells (RBCs), limiting its function to erythrocyte precursors including reticulocytes . Reticulocytes continue to produce Hb for a short period in bone marrow and peripheral circulation using residual ribosomal RNA , . Although GlyT1 has been shown to be the main contributor to glycine uptake in reticulocytes, accounting for 42% of glycine uptake in humans , the remaining glycine transfer occurs via other, less specific transporters. GlyT1 inhibition is thus not anticipated to cause complete glycine deficiency in pre-erythrocytes, which is supported by our dataset presented. Disorders of erythropoiesis are often associated with abnormal iron homeostasis . Although regulatory feedback mechanisms of reduced heme biosynthesis at inadequately low availability of iron are well understood, less is known about the impact of glycine restriction on hematopoiesis and iron regulation. The aim of this mechanistic study was to determine the effects of GlyT1 inhibition on erythropoiesis and iron homeostasis in rats using bitopertin (RG1678), a potent, selective, and reversible inhibitor of GlyT1 that has been well characterized in preclinical studies , . Materials and methods
Results
Discussion In this study, we investigated the role of GlyT1 on erythropoiesis and iron homeostasis in erythrocyte precursors by using bitopertin, a potent and selective inhibitor of GlyT1. We demonstrated that inhibition of GlyT1 disturbed Hb synthesis in rats, which manifested as a regenerative microcytic hypochromic anemia. This effect is clearly target related because microcytic hypochromic anemia with a similar magnitude of effect on MCH (21% reduction) was reported in GlyT1−/− mice [16], indicating a complete inhibition of GlyT1 under the selected experimental conditions. GlyT1 inhibition also resulted in the rapid development of iron-positive IBs within 24 hours after the first dose in the majority of reticulocytes, with a subsequent minimal extended manifestation in erythrocytes (siderocytes). Because rat reticulocytes have a bone marrow maturation phase of about 20 hours before release into circulation [17], the rapid onset of IBs strongly suggests that IB formation occurred within this maturation phase in the bone marrow. IBs were electron-microscopically characterized as iron-containing polymorphic mitochondrial remnants, indicating the persistence of uncommitted mitochondrial iron reticulocytes when heme biosynthesis is inhibited. This ultrastructure was comparable to IBs typically found in erythrocytes and erythroblasts of various forms of sideroblastic anemia [18]. In humans, acquired sideroblastic anemia has significant bone marrow involvement [19] and the diagnosis is confirmed by the widespread presence of abnormal ring sideroblasts (type III) in bone marrow samples using light and electron microscopy [20]. Type III sideroblasts were, however, absent in the rat bone marrow.