Glycine Uptake Research Articles

Expression of functional inhibitory neurotransmitter transporters GlyT1, GAT-1, and GAT-3 by astrocytes of inferior colliculus and hippocampus

Neuronal inhibition is mediated by glycine and/or GABA. Inferior colliculus (IC) neurons receive glycinergic and GABAergic inputs, whereas inhibition in hippocampus (HC) predominantly relies on GABA. Astrocytes heterogeneously express neurotransmitter transporters and are expected to adapt to the local requirements regarding neurotransmitter homeostasis. Here we analyzed the expression of inhibitory neurotransmitter transporters in IC and HC astrocytes using whole-cell patch-clamp and single-cell reverse transcription-PCR. We show that most astrocytes in both regions expressed functional glycine transporters (GlyTs). Activation of these transporters resulted in an inward current (IGly) that was sensitive to the competitive GlyT1 agonist sarcosine. Astrocytes exhibited transcripts for GlyT1 but not for GlyT2. Glycine did not alter the membrane resistance (RM) arguing for the absence of functional glycine receptors (GlyRs). Thus, IGly was mainly mediated by GlyT1. Similarly, we found expression of functional GABA transporters (GATs) in all IC astrocytes and about half of the HC astrocytes. These transporters mediated an inward current (IGABA) that was sensitive to the competitive GAT-1 and GAT-3 antagonists NO711 and SNAP5114, respectively. Accordingly, transcripts for GAT-1 and GAT-3 were found but not for GAT-2 and BGT-1. Only in hippocampal astrocytes, GABA transiently reduced RM demonstrating the presence of GABAA receptors (GABAARs). However, IGABA was mainly not contaminated by GABAAR-mediated currents as RM changes vanished shortly after GABA application. In both regions, IGABA was stronger than IGly. Furthermore, in HC the IGABA/IGly ratio was larger compared to IC. Taken together, our results demonstrate that astrocytes are heterogeneous across and within distinct brain areas. Furthermore, we could show that the capacity for glycine and GABA uptake varies between both brain regions.

Glycine Induces Migration of Microglial BV-2 Cells via SNAT-Mediated Cell Swelling

Background/Aims: The neutral, non-essential amino acid glycine has manifold functions and effects under physiological and pathophysiological conditions. Besides its function as a neurotransmitter in the central nervous system, glycine also exerts immunomodulatory effects and as an osmolyte it participates in cell volume regulation. During phagocytosis, glycine contributes to (local) cell volume-dependent processes like lamellipodium formation. Similar to the expansion of the lamellipodium we assume that glycine also affects the migration of microglial cells in a cell volume-dependent manner. Methods: Mean cell volume (MCV) and cell migration were determined using flow cytometry and trans-well migration assays, respectively. Electrophysiological recordings of the cell membrane potential (V_mem) and swelling-dependent chloride (Cl^-) currents (ICl_swell, VSOR, VRAC) were performed using the whole-cell patch clamp technique. Results: In the murine microglial cell line BV-2, flow cytometry analysis revealed that glycine (5 mM) increases the MCV by ∼9%. The glycine-dependent increase in MCV was suppressed by the partial sodium-dependent neutral amino acid transporter (SNAT) antagonist MeAIB and augmented by the Cl^- current blocker DCPIB. Electrophysiological recordings showed that addition of glycine activates a Cl^- current under isotonic conditions resembling features of the swelling-activated Cl^- current (ICl_swell). The cell membrane potential (V_mem) displayed a distinctive time course after glycine application; initially, glycine evoked a rapid depolarization mediated by Na⁺-coupled glycine uptake via SNAT, followed by a further gradual depolarization, which was fully suppressed by DCPIB. Interestingly, glycine significantly increased migration of BV-2 cells, which was suppressed by MeAIB, suggesting that SNAT is involved in the migration process of microglial cells. Conclusion: We conclude that glycine acts as a chemoattractant for microglial cells presumably by a cell volume-dependent mechanism involving SNAT-mediated cell swelling.

Elevated CO2 levels enhance the uptake and metabolism of organic nitrogen.

The effects of elevated CO2 (eCO2 ) on the relative uptake of inorganic and organic nitrogen (N) are unclear. The uptake of different N sources by pak choi (Brassica chinensis L.) seedlings supplied with a mixture of nitrate, glycine and ammonium was studied using 15 N-labelling under ambient CO2 (aCO2 ) (350 ppm) or eCO2 (650 ppm) conditions. 15 N-labelled short-term uptake and 15 N-gas chromatography mass spectrometry (GC-MS) were applied to measure the effects of eCO2 on glycine uptake and metabolism. Elevated CO2 increased the shoot biomass by 36% over 15 days, but had little effect on root growth. Over the same period, the N concentrations of shoots and roots were decreased by 30 and 2%, respectively. Elevated CO2 enhanced the uptake and N contribution of glycine, which accounted for 38-44% and 21-40% of total N uptake in roots and shoots, respectively, while the uptake of nitrate and ammonium was reduced. The increased glycine uptake resulted from the enhanced active uptake and enhanced metabolism in the roots. We conclude that eCO2 may increase the uptake and contribution of organic N forms to total plant N nutrition. Our findings provide new insights into plant N regulation under eCO2 conditions.

A glycine transporter 2-Cre knock-in mouse line for glycinergic neuron-specific gene manipulation

Glycine is an inhibitory neurotransmitter in the brainstem and spinal cord. Glycine transporter 2 (GLYT2) is responsible for the uptake of extracellular glycine. GLYT2 is specifically expressed in glycinergic neurons and thus has been used as a marker of glycinergic neurons. Here, we generated GLYT2 promotor-driven Cre recombinase (Cre)-expressing mice (GLYT2-Cre knock-in mice) to develop a tool for manipulating gene expression in glycinergic neurons. Cre activity was examined by crossing the GLYT2-Cre knock-in mice with a Cre reporter mouse line, R26R, which express β-galactosidase (β-gal) in a Cre-dependent manner. X-gal staining of GLYT2-Cre/R26R double transgenic mouse brains and spinal cords revealed that the Cre activity was primarily distributed in the brainstem, cerebellum, and spinal cord. These areas are rich in glycinergic neurons. Furthermore, we performed immunohistochemistry for β-gal combined with in situ hybridization for GLYT2 in the GLYT2-Cre/R26R double transgenic mouse brains to determine whether Cre activity is specifically localized to glycinergic neurons. The β-gal protein and GLYT2 mRNAs were colocalized in the cerebellar Golgi cells, dorsal cochlear nucleus, gigantocellular reticular nucleus, spinal trigeminal nucleus, nucleus of the trapezoid body, and lateral lemniscus. More than 98% of the GLYT2 mRNA-expressing cells in these brain regions also expressed β-gal, whereas 90–98% of the β-gal-positive cells expressed the GLYT2 mRNAs. Thus, Cre activity is specifically localized to glycinergic neurons with high fidelity in the GLYT2-Cre knock-in mice. The GLYT2-Cre knock-in mouse line will be a useful tool for studying glycinergic neurons and neurotransmission.

Effects of soil pH on the competitive uptake of amino acids by maize and microorganisms

Organic nitrogen can play an important role in plant growth, and soil pH changed greatly due to the over-use of chemical fertilizers, but the effects of soil pH on the competitive uptake of amino acids by plants and rhizosphere microorganisms are lack of detailed research. To study the effects of soil pH on the uptake of amino acids by maize and soil microorganisms, two soils from Hangzhou and Tieling were selected, and the soil pH was changed by the electrokinesis, then the 15N-labeled glycine was injected to the centrifuge tube with a short-term uptake of 4 h. Soil pH had a significant effect on the shoot and root biomass, and the optimal pH for maize shoot growth was 6.48 for Hangzhou red soil, while it was 7.65 for Tieling brown soil. For Hangzhou soil, the 15N abundance of maize shoots under pH=6.48 was significantly higher than under other treatments, and the uptake amount of 15N-glycine was also much higher. However, the 15N abundance of maize shoots and roots under pH=7.65 Tieling soil was significantly lower than it under pH=5.78, but the uptake amount of 15N-glycine under pH=7.65 was much higher. The microbial biomass C was much higher in pH=6.48 Hangzhou soil, while it was much lower in pH=7.65 Tieling soil. According to the results of root uptake, root to shoot transportation, and the competition with microorganisms, we suggested that although facing the fierce competition with microorganisms, the maize grown in pH=6.48 Hangzhou soil increased the uptake of glycine by increasing its root uptake and root to shoot transportation. While in pH=7.65 Tieling soil, the activity of microorganisms was decreased, which decreased the competition with maize for glycine, and increased the uptake of glycine by maize.

Effects of external supplied sucrose on the uptake and metabolism of glycine by pakchoi (Brassica chinensis L.)

Sucrose is crucial for plant growth, but research about the effect of sucrose on the uptake of different nitrogen (N) and the metabolism of glycine is lacking. The uptake of glycine by pakchoi, when it was in a mixture of glycine, nitrate, and ammonium or acted as the single N source under different sucrose levels were detected in a sterilized environment. The optimal sucrose level for pakchoi growth varied with N supply; it was 6 μM in the single N source of glycine, while it was 15 μM in the mixture. The N contribution of glycine increased under the optimal sucrose, while the N contribution of ammonium decreased. The effect of exogenously supplied sucrose on the uptake and metabolism of glycine is dependent on the N supply. With the single N source, the metabolism of glycine to serine in roots rather than uptake was the limiting step under a high sucrose level (300 μM). In the mixed nitrogen, active uptake and the metabolism of glycine to serine are the limiting steps under high sucrose level. Externally supplied sucrose affects the absorption and metabolism of glycine by pakchoi greatly, and this effect varied with nitrogen supply.

The fate of 13C15N labelled glycine in permafrost and surface soil at simulated thaw in mesocosms from high arctic and subarctic ecosystems

Nutrient distribution and carbon fluxes upon spring thaw are compared in mesocosms from high arctic and subarctic ecosystems dominated by Cassiope tetragona or Salix hastata/Salix arctica, in order to evaluate the possibility of plant and microbial utilization of an organic compound in thawing permafrost and surface soil. Double labeled glycine (13C15N) was added to soil columns with vegetation and to permafrost. During thaw conditions ecosystem respiration 13C was measured and 13C and 15N distribution in the ecosystem pools was quantified one day and one month after glycine addition. Near-surface soil microbes were more efficient in the uptake of intact glycine immediately upon thaw than plants. After one month plants had gained more 15N whereas microbes seemed to lose 15N originating from glycine. We observed a time lag in glycine degradation upon permafrost thaw, in contrast to surface soil thaw. Our results suggest that both arctic plants and microorganisms acquire amino acids released upon spring and permafrost thaw. Despite indications of more efficient utilization of added substrate in the High Arctic than the Subarctic, we conclude that patterns of nutrient distribution are similar and predictions based on subarctic data valid for high arctic settings.

Organic and inorganic nitrogen uptake by 21 dominant tree species in temperate and tropical forests.

Evidence shows that many tree species can take up organic nitrogen (N) in the form of free amino acids from soils, but few studies have been conducted to compare organic and inorganic N uptake patterns in temperate and tropical tree species in relation to mycorrhizal status and successional state. We labeled intact tree roots by brief 15N exposures using field hydroponic experiments in a temperate forest and a tropical forest in China. A total of 21 dominant tree species were investigated, 8 in the temperate forest and 13 in the tropical forest. All investigated tree species showed highest uptake rates for NH4+ (ammonium), followed by glycine and NO3- (nitrate). Uptake of NH4+ by temperate trees averaged 12.8 μg N g-1 dry weight (d.w.) root h-1, while those by tropical trees averaged 6.8 μg N g-1 d.w. root h-1. Glycine uptake rates averaged 3.1 μg N g-1 d.w. root h-1 for temperate trees and 2.4 μg N g-1 d.w. root h-1 for tropical trees. NO3- uptake was the lowest (averaging 0.8 μg N g-1 d.w. root h-1 for temperate trees and 1.2 μg N g-1 d.w. root h-1 for tropical trees). Uptake of NH4+ accounted for 76% of the total uptake of all three N forms in the temperate forest and 64% in the tropical forest. Temperate tree species had similar glycine uptake rates as tropical trees, with the contribution being slightly lower (20% in the temperate forest and 23% in the tropical forest). All tree species investigated in the temperate forest were ectomycorrhizal and all species but one in the tropical forest were arbuscular mycorrhizal (AM). Ectomycorrhizal trees showed significantly higher NH4+ and lower NO3- uptake rates than AM trees. Mycorrhizal colonization rates significantly affected uptake rates and contributions of NO3- or NH4+, but depended on forest types. We conclude that tree species in both temperate and tropical forests preferred to take up NH4+, with organic N as the second most important N source. These findings suggest that temperate and tropical forests demonstrate similar N uptake patterns although they differ in physiology of trees and soil biogeochemical processes.

Mouse Oocytes Acquire Mechanisms That Permit Independent Cell Volume Regulation at the End of Oogenesis.

Mouse embryos employ a unique mechanism of cell volume regulation in which glycine is imported via the GLYT1 transporter to regulate intracellular osmotic pressure. Independent cell volume regulation normally becomes active in the oocyte after ovulation is triggered. This involves two steps: the first is the release of the strong adhesion between the oocyte and zona pellucida (ZP) while the second is the activation of GLYT1. In fully-grown oocytes, release of adhesion and GLYT1 activation also occur spontaneously in oocytes removed from the follicle. It is unknown, however, whether the capacity to release oocyte-ZP adhesion or activate GLYT1 first arises in the oocyte after ovulation is triggered or instead growing oocytes already possess these capabilities but they are suppressed in the follicle. Here, we assessed when during oogenesis oocyte-ZP adhesion can be released and when GLYT1 can be activated, with adhesion assessed by an osmotic assay and GLYT1 activity determined by [3 H]-glycine uptake. Oocyte-ZP adhesion could not be released by growing oocytes until they were nearly fully grown. Similarly, the amount of GLYT1 activity that can be elicited in oocytes increased sharply at the end of oogenesis. The SLC6A9 protein that is responsible for GLYT1 activity and Slc6a9 transcripts are present in growing oocytes and increased over the course of oogenesis. Furthermore, SLC6A9 becomes localized to the oocyte plasma membrane as the oocyte grows. Thus, oocytes acquire the ability to regulate their cell volume by releasing adhesion to the ZP and activating GLYT1 as they approach the end of oogenesis. J. Cell. Physiol. 232: 2436-2446, 2017. © 2016 Wiley Periodicals, Inc.

Selective uptake of nitrogen by Suaeda salsa under drought and salt stresses and nitrogen fertilization using 15N

Plant nitrogen uptake can be influenced by drought and salt stresses and nitrogen fertilization; however, the selective uptake of different forms of nitrogen is poorly understood. We conducted a short-term 15N-labeling experiment to investigate the preferences of Suaeda salsa for nitrate, ammonium and amino acid. Our results indicated that nitrate and glycine, especially nitrate, were major nitrogen sources and glycine was absorbed after decompostion. Drought and salt stresses had no significant effect on the uptake of ammonium (p>0.05). However, nitrate uptake was strongly related to water content and nitrogen application (p<0.05) instead of salinity (p>0.05). Drought and nitrogen application reduced the uptake of nitrate by almost 65.73% and 90.29%, respectively, suggesting appropriate soil water content and nitrogen deficiency could promote nitrate uptake. The uptake of glycine was mainly influenced by water content, and drought stress decreased glycine uptake by almost 89.74%.

Effects of glucose on the uptake and metabolism of glycine in pakchoi (Brassica chinensis L.) exposed to various nitrogen sources

BackgroundPlants can absorb amino acids as a nitrogen (N) source, and glucose is an important part of root rhizodeposition and the soil sugar pool, which participates in the regulation of plant growth and uptake. In pakchoi, the effect of glucose concentration on the glycine N uptake from a nutrient mixture composed of glycine, ammonium, and nitrate, or from a single N solution of glycine alone was studied using specific substrate 15N-labeling and 15N-gas chromatography mass spectrometry.ResultsThe optimal glucose concentration for plant growth was 4.5 μM or 25 μM when supplied with glycine alone or the N mixture, respectively, and resulted in a >25% increase in seedling biomass. The addition of glucose affected the relative contribution from organic or inorganic sources to overall N uptake. When glucose was added at optimal concentrations, glycine was preferentially used as an N source, while the relative contribution from nitrate was reduced. The limiting step for glycine N contribution was active uptake in the roots in high glucose and single-N-source conditions; however, root metabolism of glycine to serine was limiting in high-glucose and mixed-N-source conditions.ConclusionsThe addition of low concentrations of glucose increased the relative uptake of organic nitrogen and reduced the uptake of nitrate, suggesting a feasible way to decrease nitrate content and increase the edible quality of vegetables.

Hexavalent chromium stress enhances the uptake of nitrate but reduces the uptake of ammonium and glycine in pak choi (Brassica chinensis L.)

Chromium (Cr) pollution affects plant growth and biochemical processes, so, the relative uptake of glycine, nitrate, and ammonium by pak choi (Brassica chinensis) seedlings in treatments with 0mgL−1 and 10mgL−1 Cr (VI) were detected by substrate-specific 15N-labelling in a sterile environment. The short-term uptake of 15N-labelled sources and 15N-enriched amino acids were detected by gas chromatography mass spectrometry to explore the mechanism by which Cr stress affects glycine uptake and metabolism, which showing that Cr stress hindered the uptake of ammonium and glycine but increased significantly the uptake of nitrate. Cr stress did not decrease the active or passive uptake of glycine, but it inhibited the conversion of glycine to serine in pak choi roots, indicating that the metabolism of glycine to serine in roots, rather than the root uptake, was the limiting step in glycine contribution to total N uptake in pak choi. Since Cr affects the relative uptake of different N sources, a feasible way to reduce Cr-induced stress is application of selective fertilization, in particular nitrate, in pak choi cultivation on Cr-polluted soil.

Effects of cadmium stress on pakchoi (Brassica chinensis L.) growth and uptake of inorganic and organic nitrogenous compounds

Exposure of cultivated plants to cadmium (Cd) can affect their growth and safety as food sources. Nitrogen (N) is important in regulating both plant growth and Cd uptake, but little research has been conducted studying the effects of Cd stress on the uptake of different forms of nitrogen. In this study, we measured the relative rates of uptake of nitrate, ammonium, and glycine by pakchoi (Brassica chinensis L.) plants under Cd stress, using substrate-specific 15N-labelling in a sterilized environment. Cd stress significantly increased the proportional contribution of nitrate in comparison with controls, while decreasing the contribution of ammonium in shoots. Overall glycine uptake decreased under Cd stress, but the proportional contribution of glycine significantly increased in roots while decreasing in shoots. The mechanism of Cd stress on glycine uptake and metabolism was detected using 15N-labelling and 15N-gas chromatography mass spectrometry (GC–MS) analysis. In shoots, Cd stress inhibited the active uptake of 15N-glycine, while enhancing the passive uptake of 15N-glycine significantly. Additionally, the short-term total uptake of glycine in shoots and roots was slightly affected by Cd stress. In comparison to controls, 15N-labelled glycine was found to be significantly higher and serine lower in Cd-stressed plants, indicating that Cd stress inhibited the conversion of glycine to serine. We posit that Cd stress affects the overall nitrogen uptake in pakchoi plants, but that an inhibition in the metabolism of glycine to serine, rather than root uptake, is the limiting step for glycine contribution.

Contributions of system A subtypes to α-methylaminoisobutyric acid uptake by placental microvillous membranes of human and rat.

System A consists of three subtypes, sodium-coupled neutral amino acid transporter 1 (SNAT1), SNAT2, and SNAT4, which are all expressed in the placenta. The aim of this study was to evaluate the contributions of each of the three subtypes to total system A-mediated uptake in placental MVM of human and rat, using betaine and L-arginine as subtype-selective inhibitors of SNAT2 and SNAT4, respectively. Appropriate concentrations of betaine and L-arginine for subtype-selective inhibition in SNAT-overexpressing cells were identified. It was found that 10mM betaine specifically and almost completely inhibited human and rat SNAT2-mediated [14C]α-methylaminoisobutyric acid ([14C]MeAIB) uptake, while 5mM L-arginine specifically and completely inhibited [3H]glycine uptake via human SNAT4, as well as [14C]MeAIB uptake via rat SNAT4. In both human and rat placental MVM vesicles, sodium-dependent uptake of [14C]MeAIB was almost completely inhibited by 20mM unlabeled MeAIB. L-Arginine (5mM) partly inhibited the uptake in humans, but hardly affected that in rats. Betaine (10mM) partly inhibited the uptake in rats, but hardly affected it in humans. These results suggest that SNAT1 is most likely the major contributor to system A-mediated MeAIB uptake by human and rat MVM vesicles and that the remaining uptake is mainly mediated by SNAT4 in humans and SNAT2 in rats. Thus, inhibition studies using betaine and L-arginine are useful to characterize the molecular mechanisms of system A-mediated transport.

Assessment of a glycine uptake inhibitor in animal models of effort-related choice behavior: implications for motivational dysfunctions.

Motivated behavior can be characterized by a substantial exertion of effort, and organisms often make effort-related decisions based upon analyses of work-related response costs and reinforcement preference. Moreover, alterations in effort-based choice can be seen in people with major depression and schizophrenia. Effort-related decision making is studied using tasks offering choices between high effort options leading to highly valued reinforces vs low effort/low reward options. Interference with dopamine (DA) transmission by administration of the DA D2 family antagonist haloperidol biases behavior towards the lower effort option that can be obtained with minimal work, and previous research has shown that DA interacts with other transmitters, including adenosine and GABA, to regulate effort-based choice. The present studies focused upon the ability of the glycine transport inhibitor bitopertin to attenuate haloperidol-induced shifts in effort-related choice behavior. Effort-based choice in rats was assessed using the concurrent fixed ratio (FR) 5/chow feeding choice task and the T-maze barrier choice procedure. Haloperidol shifted effort-based choice, biasing animals towards the low effort option in each task. Co-administration of bitopertin (1.0-10.0mg/kg) significantly attenuated haloperidol-induced shifts in choice behavior, but the same doses of bitopertin had no effect when administered alone. These results indicated that elevation of extracellular glycine via inhibition of glycine uptake was able to reverse the effects of D2 antagonism. Increases in extracellular glycine, possibly through actions on the glycine allosteric site on the NMDA receptor, may be a useful strategy for treating motivational dysfunctions in humans.

Endogenous expression of the glycine transporter 2 in C6 glioma cells: a preliminary study

C6 glioma cells have been proposed as a cell model system to study regulation of endogenously-expressed glycine transporter 1 (GlyT1). However, there are no reports demonstrating expression of glycine transporter 2 (GlyT2) in this cell line. To explore this possibility, immunoprecipitation, western blotting, confocal microscopy, as well as glycine uptake assays using selective inhibitors to GlyT1 or GlyT2 were employed. Glycine uptake assays using a selective inhibitor against GlyT2 provided evidence that this transporter could also be expressed in this cell line. Immunoprecipitation, immunoblotting, and confocal microscopic analysis also showed presence of both GlyT1 and GlyT2 in this cell line. Taken together, results provide evidence that in addition to GlyT1, GlyT2 is also endogenously expressed in C6 glioma cells. Results presented here will help researchers to carefully evaluate use of this cell line in studies involving GlyT1.

Effect of pH on the uptake and metabolism of glycine in pak choi (Brassica chinensis L.)

Although plants can utilize amino acids as a nitrogen (N) source, it is unclear how pH affects the uptake of amino acids. We investigated the relative uptake of glycine, nitrate, and ammonium by pak choi at different pH levels using specific substrate 15N-labelling in a sterile environment. Further, the mechanisms underlying the influence of pH on glycine uptake and metabolism were examined by 15N-labelling short-term uptake and 15N-gas chromatography–mass spectrometry analysis. The optimal pH for glycine uptake was 7.0, for both single and mixed nitrogen sources, whereas that for pak choi growth (mixedN sources) was 6.2. Glycine served as an important N source in the sterile environment, accounting for 19.0%–33.1% of N in the mixed N sources, and contribution peaked at pH 7.0. Little 15N-labelled glycine was detected in shoots, indicating that the majority of the glycine absorbed by the roots was metabolized to other amino acids. The limiting step for N contribution by glycine was root uptake at lower pH levels, particularly active uptake, and root metabolism of glycine to serine at pH 8.0. These results indicate that pak choi can efficiently utilize glycine, and that pH has a substantial effect on glycine uptake and metabolism.

Genome Sequence of the Arsenic-Resistant Haladaptatus sp. Strain R4 Isolated from Ramnagar, West Bengal, India

Here, we present the draft genome of Haladaptatus sp. strain R4, a halophilic archaea that produces an orange-pink pigment and is capable of growing in a wide salinity range. The genome assembly shows genes for arsenic resistance, siderophore production, trehalose and glycine betaine biosynthesis, uptake and transporters of sodium, potassium, and chloride ions.

Characterization of Human and Yeast Mitochondrial Glycine Carriers with Implications for Heme Biosynthesis and Anemia

Heme is an essential molecule in many biological processes, such as transport and storage of oxygen and electron transfer as well as a structural component of hemoproteins. Defects of heme biosynthesis in developing erythroblasts have profound medical implications, as represented by sideroblastic anemia. The synthesis of heme requires the uptake of glycine into the mitochondrial matrix where glycine is condensed with succinyl coenzyme A to yield δ-aminolevulinic acid. Herein we describe the biochemical and molecular characterization of yeast Hem25p and human SLC25A38, providing evidence that they are mitochondrial carriers for glycine. In particular, the hem25Δ mutant manifests a defect in the biosynthesis of δ-aminolevulinic acid and displays reduced levels of downstream heme and mitochondrial cytochromes. The observed defects are rescued by complementation with yeast HEM25 or human SLC25A38 genes. Our results identify new proteins in the heme biosynthetic pathway and demonstrate that Hem25p and its human orthologue SLC25A38 are the main mitochondrial glycine transporters required for heme synthesis, providing definitive evidence of their previously proposed glycine transport function. Furthermore, our work may suggest new therapeutic approaches for the treatment of congenital sideroblastic anemia.

Effects of GlyT1 inhibition on erythropoiesis and iron homeostasis in rats.

Glycine is a key rate-limiting component of heme biosynthesis in erythropoietic cells, where the high intracellular glycine demand is primarily supplied by the glycine transporter 1 (GlyT1). The impact of intracellular glycine restriction after GlyT1 inhibition on hematopoiesis and iron regulation is not well established. We investigated the effects of a potent and selective inhibitor of GlyT1, bitopertin, on erythropoiesis and iron homeostasis in rats. GlyT1 inhibition significantly affected erythroid heme biosynthesis, manifesting as microcytic hypochromic regenerative anemia with a 20% steady-state reduction in hemoglobin. Reduced erythropoietic iron utilization was characterized by down-regulation of the transferrin receptor 1 (TfR1) on reticulocytes and modest increased iron storage in the spleen. Hepatic hepcidin expression was not affected. However, under the condition of reduced heme biosynthesis with reduced iron reutilization and increased storage iron, hepcidin at the lower and higher range of normal showed a striking role in tissue distribution of iron. Rapid formation of iron-positive inclusion bodies (IBs) was observed in circulating reticulocytes, with an ultrastructure of iron-containing polymorphic mitochondrial remnants. IB or mitochondrial iron accumulation was absent in bone marrow erythroblasts. In conclusion, GlyT1 inhibition in rats induced a steady-state microcytic hypochromic regenerative anemia and a species-specific accumulation of uncommitted mitochondrial iron in reticulocytes. Importantly, this glycine-restricted anemia provides no feedback signal for increased systemic iron acquisition and the effects reported are pathogenetically distinct from systemic iron-overload anemias and erythropoietic disorders such as acquired sideroblastic anemia.