Inhibition of sodium-glucose cotransporter 2 ameliorates renal injury in a novel medaka model of nonalcoholic steatohepatitis-related kidney disease.

The effect of sodium-glucose cotransporter 2 inhibitor (SGLT2I) on nonalcoholic steatohepatitis (NASH) has been reported, but there are few studies on its effect on NASH-related renal injury. In this study, we examined the effect of SGLT2I using a novel medaka fish model of NASH-related kidney disease, which was developed by feeding the d-rR/Tokyo strain a high-fat diet. SGLT2I was administered by dissolving it in water of the feeding tank. SGLT2I ameliorates macrophage accumulation and oxidative stress and maintained mitochondrial function in the kidney. The results demonstrate the effect of SGLT2I on NASH-related renal injury and the usefulness of this novel animal model for research into NASH-related complications.

NLRX1 Enhances Glutamate Uptake and Inhibits Glutamate Release by Astrocytes.

Uptake of glutamate from the extracellular space and glutamate release to neurons are two major processes conducted by astrocytes in the central nervous system (CNS) that protect against glutamate excitotoxicity and strengthen neuronal firing, respectively. During inflammatory conditions in the CNS, astrocytes may lose one or both of these functions, resulting in accumulation of the extracellular glutamate, which eventually leads to excitotoxic neuronal death, which in turn worsens the CNS inflammation. NLRX1 is an innate immune NOD-like receptor that inhibits the major inflammatory pathways. It is localized in the mitochondria and was shown to inhibit cell death, enhance ATP production, and dampen oxidative stress. In the current work, using primary murine astrocyte cultures from WT and Nlrx1-/- mice, we demonstrate that NLRX1 potentiates astrocytic glutamate uptake by enhancing mitochondrial functions and the functional activity of glutamate transporters. Also, we report that NLRX1 inhibits glutamate release from astrocytes by repressing Ca2+-mediated glutamate exocytosis. Our study, for the first time, identified NLRX1 as a potential regulator of glutamate homeostasis in the CNS.

Feedback adaptation of synaptic excitability via Glu:Na+ symport driven astrocytic GABA and Gln release.

Glutamatergic transmission composed of the arriving of action potential at the axon terminal, fast vesicular Glu release, postsynaptic Glu receptor activation, astrocytic Glu clearance and Glu→Gln shuttle is an abundantly investigated phenomenon. Despite its essential role, however, much less is known about the consequences of the mechanistic connotations of Glu:Na+ symport. Due to the coupled Na+ transport, Glu uptake results in significantly elevated intracellular astrocytic [Na+] that markedly alters the driving force of other Na+-coupled astrocytic transporters. The resulting GABA and Gln release by reverse transport through the respective GAT-3 and SNAT3 transporters help to re-establish the physiological Na+ homeostasis without ATP dissipation and consequently leads to enhanced tonic inhibition and replenishment of axonal glutamate pool. Here, we place this emerging astrocytic adjustment of synaptic excitability into the centre of future perspectives. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

Excitatory amino acid transporters: recent insights into molecular mechanisms, novel modes of modulation and new therapeutic possibilities.

The five excitatory amino acid transporters (EAAT1-5) mediating the synaptic uptake of the major excitatory neurotransmitter glutamate are differently expressed throughout the CNS and at the synaptic level. Although EAATs are crucial for normal excitatory neurotransmission, explorations into the physiological functions mediated by the different transporter subtypes and their respective therapeutic potential have so far been sparse, in no small part due to the limited selection of pharmacological tools available. In the present update, we outline important new insights into the molecular compositions of EAATs and their intricate transport process, the novel approaches to pharmacological modulation of the transporters that have emerged, and interesting new perspectives in EAAT as drug targets proposed in recent years.

Excitotoxicity triggered by neonatal monosodium glutamate treatment and blood-brain barrier function.

It is likely that monosodium glutamate (MSG) is the excitotoxin that has been most commonly employed to characterize the process of excitotoxicity and to improve understanding of the ways that this process is related to several pathological conditions of the central nervous system. Excitotoxicity triggered by neonatal MSG treatment produces a significant pathophysiological impact on adulthood, which could be due to modifications in the blood-brain barrier (BBB) permeability and vice versa. This mini-review analyzes this topic through brief descriptions about excitotoxicity, BBB structure and function, role of the BBB in the regulation of Glu extracellular levels, conditions that promote breakdown of the BBB, and modifications induced by neonatal MSG treatment that could alter the behavior of the BBB. In conclusion, additional studies to better characterize the effects of neonatal MSG treatment on excitatory amino acids transporters, ionic exchangers, and efflux transporters, as well as the role of the signaling pathways mediated by erythropoietin and vascular endothelial growth factor in the cellular elements of the BBB, should be performed to identify the mechanisms underlying the increase in neurovascular permeability associated with excitotoxicity observed in several diseases and studied using neonatal MSG treatment.

Excitatory amino acid transporters: roles in glutamatergic neurotransmission.

Excitatory amino acid transporters or EAATs are the major transport mechanism for extracellular glutamate in the nervous system. This family of five carriers not only displays an impressive ability to regulate ambient extracellular glu concentrations but also regulate the temporal and spatial profile of glu after vesicular release. This dynamic form of regulation mediates several characteristic of synaptic, perisynaptic, and spillover activation of ionotropic and metabotropic receptors. EAATs function through a secondary active, electrogenic process but also possess a thermodynamically uncoupled ligand gated anion channel activity, both of which have been demonstrated to play a role in regulation of cellular activity. This review will highlight the inception of EAATs as a focus of research, the transport and channel functionality of the carriers, and then describe how these properties are used to regulate glutamatergic neurotransmission.

Mechanisms of glutamate transport.

L-Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system and plays important roles in a wide variety of brain functions, but it is also a key player in the pathogenesis of many neurological disorders. The control of glutamate concentrations is critical to the normal functioning of the central nervous system, and in this review we discuss how glutamate transporters regulate glutamate concentrations to maintain dynamic signaling mechanisms between neurons. In 2004, the crystal structure of a prokaryotic homolog of the mammalian glutamate transporter family of proteins was crystallized and its structure determined. This has paved the way for a better understanding of the structural basis for glutamate transporter function. In this review we provide a broad perspective of this field of research, but focus primarily on the more recent studies with a particular emphasis on how our understanding of the structure of glutamate transporters has generated new insights.

The SLC1 high-affinity glutamate and neutral amino acid transporter family.

Glutamate transporters play important roles in the termination of excitatory neurotransmission and in providing cells throughout the body with glutamate for metabolic purposes. The high-affinity glutamate transporters EAAC1 (SLC1A1), GLT1 (SLC1A2), GLAST (SLC1A3), EAAT4 (SLC1A6), and EAAT5 (SLC1A7) mediate the cellular uptake of glutamate by the co-transport of three sodium ions (Na(+)) and one proton (H(+)), with the counter-transport of one potassium ion (K(+)). Thereby, they protect the CNS from glutamate-induced neurotoxicity. Loss of function of glutamate transporters has been implicated in the pathogenesis of several diseases, including amyotrophic lateral sclerosis and Alzheimer's disease. In addition, glutamate transporters play a role in glutamate excitotoxicity following an ischemic stroke, due to reversed glutamate transport. Besides glutamate transporters, the SLC1 family encompasses two transporters of neutral amino acids, ASCT1 (SLC1A4) and ASCT2 (SLC1A5). Both transporters facilitate electroneutral exchange of amino acids in neurons and/or cells of the peripheral tissues. Some years ago, a high resolution structure of an archaeal homologue of the SLC1 family was determined, followed by the elucidation of its structure in the presence of the substrate aspartate and the inhibitor d,l-threo-benzyloxy aspartate (d,l-TBOA). Historically, the first few known inhibitors of SLC1 transporters were based on constrained glutamate analogs which were active in the high micromolar range but often also showed off-target activity at glutamate receptors. Further development led to the discovery of l-threo-ß-hydroxyaspartate derivatives, some of which effectively inhibited SLC1 transporters at nanomolar concentrations. More recently, small molecule inhibitors have been identified whose structures are not based on amino acids. Activators of SLC1 family members have also been discovered but there are only a few examples known.

Molecular determinants for functional differences between alanine-serine-cysteine transporter 1 and other glutamate transporter family members.

The ASCTs (alanine, serine, and cysteine transporters) belong to the solute carrier family 1 (SLC1), which also includes the human glutamate transporters (excitatory amino acid transporters, EAATs) and the prokaryotic aspartate transporter GltPh. Despite the high degree of amino acid sequence identity between family members, ASCTs function quite differently from the EAATs and GltPh. The aim of this study was to mutate ASCT1 to generate a transporter with functional properties of the EAATs and GltPh, to further our understanding of the structural basis for the different transport mechanisms of the SLC1 family. We have identified three key residues involved in determining differences between ASCT1, the EAATs and GltPh. ASCT1 transporters containing the mutations A382T, T459R, and Q386E were expressed in Xenopus laevis oocytes, and their transport and anion channel functions were investigated. A382T and T459R altered the substrate selectivity of ASCT1 to allow the transport of acidic amino acids, particularly l-aspartate. The combination of A382T and T459R within ASCT1 generates a transporter with a similar profile to that of GltPh, with preference for l-aspartate over l-glutamate. Interestingly, the amplitude of the anion conductance activated by the acidic amino acids does not correlate with rates of transport, highlighting the distinction between these two processes. Q386E impaired the ability of ASCT1 to bind acidic amino acids at pH 5.5; however, this was reversed by the additional mutation A382T. We propose that these residues differences in TM7 and TM8 combine to determine differences in substrate selectivity between members of the SLC1 family.

[Brain repair after ischemic stroke: role of neurotransmitters in post-ischemic neurogenesis]. / Neurorrestauración tras la isquemia cerebral: papel de los neurotransmisores en la neurogénesis postisquémica.

INTRODUCTION: Brain ischemia and reperfusion produce alterations in the microenvironment of the parenchyma, including ATP depletion, ionic homeostasis alterations, inflammation, release of multiple cytokines and abnormal release of neurotransmitters. As a consequence, the induction of proliferation and migration of neural stem cells towards the peri-infarct region occurs. DEVELOPMENT: The success of new neurorestorative treatments for damaged brain implies the need to know, with greater accuracy, the mechanisms in charge of regulating adult neurogenesis, both under physiological and pathological conditions. Recent evidence demonstrates that many neurotransmitters, glutamate in particular, control the subventricular zone, thus being part of the complex signalling network that influences the production of new neurons. CONCLUSION: Neurotransmitters provide a link between brain activity and subventricular zone neurogenesis. Therefore, a deeper knowledge of the role of neurotransmitters systems, such as glutamate and its transporters, in adult neurogenesis, may provide a valuable tool to be used as a neurorestorative therapy in this pathology.

The mode of retinal presynaptic inhibition switches with light intensity.

Excitatory amino acid transporters (EAATs) terminate signaling in the CNS by clearing released glutamate. Glutamate also evokes an EAAT-mediated Cl(-) current, but its role in CNS signaling is poorly understood. We show in mouse retina that EAAT-mediated Cl(-) currents that were evoked by light inhibit rod pathway signaling. EAATs reside on rod bipolar cell axon terminals where GABA and glycine receptors also mediate light-evoked inhibition. We found that the mode of inhibition depended on light intensity. Dim light evoked GABAergic and glycinergic inhibition with rapid kinetics and a large spatial extent. Bright light evoked predominantly EAAT-mediated inhibition with slow kinetics and a small spatial extent. The switch to EAAT-mediated signaling in bright light supplements receptor-mediated signaling to expand the dynamic range of inhibition and contributes to the transition from rod to cone signaling by suppressing rod pathway signaling in bright light conditions.

[Roles of ATP receptors in the regulation of various functions in spinal microglia].

It is shown that glial cells have a pivotal influence on the formation of neuronal network in central nerve system. Moreover, spinal microglia has some important roles in the development and progression of various neurological disorders. Therefore, it is possible that modulation of microglial activity may be sufficient to alleviate those harmful responses. ATP is one of signaling molecules in the spinal cord, and involved in regulation of several microglial functions through the binding of P2X and P2Y receptors. Thus, I focused on the ATP-mediated regulation mechanisms for the two important proteins, which are p38 MAP kinase and excitatory amino acid transporters (EAATs), in cultured spinal microglia. Mounting evidence indicates that p38 in spinal microglia has crucial roles in some neurological diseases. Furthermore, it is recently suggested that microglial EAATs might participate in the homeostasis of glutamate in synapses. This review summarizes our finding regarding the involvement of P2Y receptors and ß-adrenergic receptors in the regulation of p38 phosphorylation, and the mechanism of P2X7 receptor-mediated downregulation of EAATs function.

18F-fluorodeoxyglucose uptake in tumor.

The 18fluorine-fluorodeoxyglucose (18F-FDG), a glucose analog, has been widely used in tumor imaging. The tumoral uptake of 18F-FDG is based upon enhanced glycolysis. Following administration, 18F-FDG is phosphorylated and trapped intracellularly that forms the basis of PET imaging. An important mechanism to transport 18F-FDG into the tumor cell is based upon the action of glucose transporter proteins; furthermore, highly active hexokinase bound to tumor mitochondria helps to trap 18F-FDG into the cell. In addition, enhanced 18F-FDG uptake may be due to relative hypoxia in tumor masses, which activates the anaerobic glycolytic pathway. In spite of these processes, 18F-FDG uptake is relatively nonspecific since all living cells need glucose. Clinical application of 18F-FDG imaging is therefore recommended in carefully selected patients.

The glial glutamate transporter 1 (GLT1) is expressed by pancreatic beta-cells and prevents glutamate-induced beta-cell death.

Glutamate is the major excitatory neurotransmitter of the central nervous system (CNS) and may induce cytotoxicity through persistent activation of glutamate receptors and oxidative stress. Its extracellular concentration is maintained at physiological concentrations by high affinity glutamate transporters of the solute carrier 1 family (SLC1). Glutamate is also present in islet of Langerhans where it is secreted by the α-cells and acts as a signaling molecule to modulate hormone secretion. Whether glutamate plays a role in islet cell viability is presently unknown. We demonstrate that chronic exposure to glutamate exerts a cytotoxic effect in clonal ß-cell lines and human islet ß-cells but not in α-cells. In human islets, glutamate-induced ß-cell cytotoxicity was associated with increased oxidative stress and led to apoptosis and autophagy. We also provide evidence that the key regulator of extracellular islet glutamate concentration is the glial glutamate transporter 1 (GLT1). GLT1 localizes to the plasma membrane of ß-cells, modulates hormone secretion, and prevents glutamate-induced cytotoxicity as shown by the fact that its down-regulation induced ß-cell death, whereas GLT1 up-regulation promoted ß-cell survival. In conclusion, the present study identifies GLT1 as a new player in glutamate homeostasis and signaling in the islet of Langerhans and demonstrates that ß-cells critically depend on its activity to control extracellular glutamate levels and cellular integrity.

Aspectos moleculares de la encefalopatía hepática / Molecular aspects of hepatic encephalopathy

Introducción: La fibrosis hepática y su etapa final, la cirrosis, representan un enorme problema de salud mundial. La encefalopatía hepática (EH) o encefalopatía portosistémica es una afección clínica de la cirrosis a largo plazo. En esta revisión se destacan las bases moleculares de la EH, así como el papel del estrés oxidativo en el desarrollo de esta enfermedad.Fuentes: Diversos estudios señalan que la EH es de origen multifactorial, las alteraciones en la barrera hematoencefálica, sustancias como el amonio y el manganeso, así como alteraciones en la neurotransmisión de dopamina, glutamato y GABA, se han implicado en la patogenia de esta enfermedad. Desarrollo: La EH es una complicación severa de la insuficiencia hepática tanto aguda como crónica. Neuropatológicamente, se caracteriza por cambios astrocitarios conocidos como astrocitosis Alzheimer tipo II y por la expresión alterada de proteínas específicas de astrocito, como la proteína acídica fibrilar glial, la glutamina sintetasa, los inhibidores de la monoaminooxidasa y los receptores periféricos tipo benzodiacepina.Conclusiones: La EH es un síndrome neuropsiquiátrico complejo asociado a una falla hepática. Estas alteraciones son producto de un incremento de estrés oxidativo en el cerebro como consecuencia de la acción de neurotoxinas. La principal estrategia para el tratamiento de la EH se dirige a la reducción del amonio, ya sea por la disminución de su absorción/producción o promoviendo su eliminación (AU)

Introduction: Liver fibrosis and its end stage, cirrhosis, is an enormous worldwide health problem. Hepatic encephalopathy (HE) or portal-systemic encephalopathy continues to be a major clinical problem of long-term cirrhosis. In this review we emphasise the molecular basis of HE and the involvement of oxidative stress in the development of this disease.Background: Several studies suggest that the pathogenesis of HE could be multifactorial and have implicated different factors, such as alterations in blood brain barrier, substances; such as ammonia and manganese, neurotransmission disorders such as dopamine, glutamate and GABA.Development: HE is a severe complication of both acute and chronic liver failure. Neuropathologically, it is characterized by astrocyte changes known as Alzheimer type II astrocytosis. In addition, astrocytes manifest altered expression of astrocyte-specific proteins, such as, glial fibrillary acidic protein, glutamine synthetase, monoamine oxidase and peripheral type benzodiazepine receptors. Conclusions:HE is a complex neuropsychiatric syndrome associated with liver failure. These alterations are products of increases in oxidative stress in brain due to neurotoxin activity. The main strategy for HE treatment is directed at ammonia reduction, which can be achieved either by decreasing its absorption/production or increasing its removal (AU)

The role of excitatory amino acid transporters in cerebral ischemia.

Glutamate is an excitatory neurotransmitter that plays a major role in the pathogenesis of ischemia brain injury. The regulation of glutamate neurotransmission is carried out by excitatory amino acid transporters (EAATs) that act through reuptake of glutamate into cells. EAATs may also release glutamate into the extracellular space in a calcium-independent manner during ischemia and dysfunction of EAATs is specifically implicated in the pathology of cerebral ischemia. Recent studies show that up-regulation of EAAT2 provides neuroprotection during ischemic insult. This review summarizes current knowledge regarding the role of EAATs in cerebral ischemia.

Functional importance of GGXG sequence motifs in putative reentrant loops of 2HCT and ESS transport proteins.

The 2HCT and ESS families are two families of secondary transporters. Members of the two families are unrelated in amino acid sequence but share similar hydropathy profiles, which suggest a similar folding of the proteins in membranes. Structural models show two homologous domains containing five transmembrane segments (TMSs) each, with a reentrant or pore loop between the fourth and fifth TMSs in each domain. Here we show that GGXG sequence motifs present in the putative reentrant loops are important for the activity of the transporters. Mutation of the conserved Gly residues to Cys in the motifs of the Na(+)-citrate transporter CitS in the 2HCT family and the Na(+)-glutamate transporter GltS in the ESS family resulted in strongly reduced transport activity. Similarly, mutation of the variable residue "X" to Cys in the N-terminal half of GltS essentially inactivated the transporter. The corresponding mutations in the N- and C-terminal halves of CitS reduced transport activity to 60 and 25% of that of the wild type, respectively. Residual activity of any of the mutants could be further reduced by treatment with the membrane permeable thiol reagent N-ethylmaleimide (NEM). The X to Cys mutation (S405C) in the cytoplasmic loop in the C-terminal half of CitS rendered the protein sensitive to the bulky, membrane impermeable thiol reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AmdiS) added at the periplasmic side of the membrane, providing further evidence that this part of the loop is positioned between the transmembrane segments. The putative reentrant loop in the C-terminal half of the ESS family does not contain the GGXG motif, but a conserved stretch rich in Gly residues. Cysteine-scanning mutagenesis of a stretch of 18 residues in the GltS protein revealed two residues important for function. Mutant N356C was completely inactivated by treatment with NEM, and mutant P351C appeared to be the counterpart of mutant S405C of CitS; the mutant was inactivated by AmdiS added at the periplasmic side of the membrane. The data support, in general, the structural and mechanistic similarity between the ESS and 2HCT transporters and, more particularly, the two-domain structure of the transporters and the presence and functional importance of the reentrant loops present in each domain. It is proposed that the GGXG motifs are at the vertex of the reentrant loops.

Low-affinity excitatory amino acid uptake in hippocampal astrocytes: a possible role of Na+/dicarboxylate cotransporters.

The excitatory amino acid transporters (EAATs) underlie the so-called "high affinity" uptake of glutamate, which is well characterized. In contrast, the "low-affinity" uptake of glutamate remains poorly defined, and it has been discussed whether it may represent a mere in vitro artifact. Here we have visualized "low-affinity" excitatory amino acid uptake sites by incubating rat hippocampal slices with the glutamate analogue D-aspartate in the presence of PMB-TBOA, which blocks the EAATs. After fixation of the slices, D-aspartate taken up into the tissue was localized with the use of light microscopic immunoperoxidase and electron microscopic immunogold methods, exploiting highly specific antibodies against D-aspartate. PMB-TBOA blocked uptake of both low and high exogenous D-aspartate concentrations (0.01-1.0 mM) into nerve terminals, as well as the uptake of 0.01 mM D-aspartate into astrocytes. Interestingly, there was a residual PMB-TBOA insensitive uptake of D-aspartate in astrocytes at higher exogenous D-aspartate concentrations (0.05-1.0 mM), strongly suggesting that astrocytes have "low-affinity" uptake sites for excitatory amino acid. The PMB-TBOA insensitive D-aspartate uptake in astrocytes was sodium dependent and inhibited by succinate and to certain extent by homocysteate, but not by cystine or DIDS. We suggest that excitatory amino acid is transported into astrocytes in a "low-affinity" fashion by sodium/dicarboxylate transporters.

Diabetes mellitus, part 1: physiology and complications.

In part 1 of this 2-part article the author discusses the physiology and complications of diabetes mellitus (DM), a chronic and progressive disorder which affects all ages of the population. The number of people diagnosed with diabetes is approximately 1.8 million and an estimated further 1 million are undiagnosed (Department of Health, 2005). In the UK, 1-2% of the population have diabetes and among school children this is approximately 2 in 1000 (Watkins, 1996). There are two main types of diabetes--type 1 and type 2 (Porth, 2005). The aetiology of DM is unknown; however, genetic and environmental factors have been linked to its development. Type 1 results from the loss of insulin production in the beta cells of the pancreas, and type 2 from a lack of serum insulin or poor uptake of glucose into the cells. Diabetes causes disease in many organs in the body, which may be life-threatening if untreated. Complications such as heart disease, vascular disease, renal failure and blindness (Roberts, 2005) have all been reported. The increased prevalence may be caused by factors such as environmental aspects, diet, an ageing population and low levels of physical exercise.

High-density presynaptic transporters are required for glutamate removal from the first visual synapse.

Reliable synaptic transmission depends not only on the release machinery and the postsynaptic response mechanism but also on removal or degradation of transmitter from the synaptic cleft. Accumulating evidence indicates that postsynaptic and glial excitatory amino acid transporters (EAATs) contribute to glutamate removal. However, the role of presynaptic EAATs is unclear. Here, we show in the mouse retina that glutamate is removed from the synaptic cleft at the rod to rod bipolar cell (RBC) synapse by presynaptic EAATs rather than by postsynaptic or glial EAATs. The RBC currents evoked by electrical stimulation of rods decayed slowly after pharmacological blockade of EAATs. Recordings of the evoked RBC currents from EAAT subtype-deficient mice and the EAAT-coupled anion current reveal that functional EAATs are localized to rod terminals. Model simulations suggest that rod EAATs are densely packed near the release site and that rods are equipped with an almost self-sufficient glutamate recollecting system.