Intracellular accumulation of amino acids increases synaptic potentials in rat hippocampal slices.

The application of high concentrations of taurine induces long-lasting potentiation of synaptic responses and axon excitability. This phenomenon seems to require the contribution of a transport system with a low affinity for taurine. The prototypic taurine transporter TauT (SLC6A6) was discarded by experimental evidence obtained in TauT-KO mice. The purpose of the present study was to determine whether the proton-coupled amino acid transporter 1 (PAT1; SLC36A1) which is a transport system with low affinity and high capacity for a great variety of amino acids including taurine, contributes to the taurine-induced synaptic potentiation. In rat hippocampal slices, the application of several amino acids (L- and D-alanine, L-glutamine, ß-guanidinopropionic acid, glycine, L-histidine, L- and D-serine, sarcosine, L- and D-threonine) imitated the synaptic potentiation induced by taurine. The magnitude of the potentiation caused by some of these amino acids was even greater than that induced by taurine. By contrast, the application of other amino acids (L-arginine, betaine, L-leucine, L-methionine, L- and D-proline, and L-valine) did not induce potentiation. The behaviour of these different amino acids on synaptic potentiation is not compatible with a role of PAT1 in synaptic potentiation. There was a positive correlation between the accumulation of the different amino acids in the slice and the magnitude of synaptic potentiation induced by them. Some of the amino acids inducing synaptic potentiation, like taurine and L-threonine, also increased electrical resistance of the slice, whereas L-leucine did not modify this parameter. Modifications induced by either taurine or L-threonine in synaptic potentiation, slice resistance and amino acid accumulation were dependent on extracellular chloride concentration. These findings support the idea that the accumulation of amino acids throughout the action of transporters causes cell swelling enhancing the electrical resistance of the slice, which by itself could be sufficient to increase field synaptic potentials.

Fumarate Hydratase Deletion in Pancreatic ß Cells Leads to Progressive Diabetes.

We explored the role of the Krebs cycle enzyme fumarate hydratase (FH) in glucose-stimulated insulin secretion (GSIS). Mice lacking Fh1 in pancreatic ß cells (Fh1ßKO mice) appear normal for 6-8 weeks but then develop progressive glucose intolerance and diabetes. Glucose tolerance is rescued by expression of mitochondrial or cytosolic FH but not by deletion of Hif1α or Nrf2. Progressive hyperglycemia in Fh1ßKO mice led to dysregulated metabolism in ß cells, a decrease in glucose-induced ATP production, electrical activity, cytoplasmic [Ca2+]i elevation, and GSIS. Fh1 loss resulted in elevated intracellular fumarate, promoting succination of critical cysteines in GAPDH, GMPR, and PARK 7/DJ-1 and cytoplasmic acidification. Intracellular fumarate levels were increased in islets exposed to high glucose and in islets from human donors with type 2 diabetes (T2D). The impaired GSIS in islets from diabetic Fh1ßKO mice was ameliorated after culture under normoglycemic conditions. These studies highlight the role of FH and dysregulated mitochondrial metabolism in T2D.

Taurine content in different brain structures during ageing: effect on hippocampal synaptic plasticity.

A reduction in taurine content accompanies the ageing process in many tissues. In fact, the decline of brain taurine levels has been associated with cognitive deficits whereas chronic administration of taurine seems to ameliorate age-related deficits such as memory acquisition and retention. In the present study, using rats of three age groups (young, adult and aged) we determined whether the content of taurine and other amino acids (glutamate, serine, glutamine, glycine, alanine and GABA) was altered during ageing in different brain areas (cerebellum, cortex and hippocampus) as well non-brain tissues (heart, kidney, liver and plasma). Moreover, using hippocampal slices we tested whether ageing affects synaptic function and plasticity. These parameters were also determined in aged rats fed with either taurine-devoid or taurine-supplemented diets. With age, we found heterogeneous changes in amino acid content depending on the amino acid type and the tissue. In the case of taurine, its content was reduced in the cerebellum of adult and aged rats, but it remained unchanged in the hippocampus, cortex, heart and liver. The synaptic response amplitude decreased in aged rats, although the late phase of long-term synaptic potentiation (late-LTP), a taurine-dependent process, was not altered. Our study highlights the stability of taurine content in the hippocampus during ageing regardless of whether taurine was present in the diet, which is consistent with the lack of changes detected in late-LTP. These results indicate that the beneficial effects of taurine supplementation might be independent of the replenishment of taurine stores.

GDH-Dependent Glutamate Oxidation in the Brain Dictates Peripheral Energy Substrate Distribution.

Glucose, the main energy substrate used in the CNS, is continuously supplied by the periphery. Glutamate, the major excitatory neurotransmitter, is foreseen as a complementary energy contributor in the brain. In particular, astrocytes actively take up glutamate and may use it through oxidative glutamate dehydrogenase (GDH) activity. Here, we investigated the significance of glutamate as energy substrate for the brain. Upon glutamate exposure, astrocytes generated ATP in a GDH-dependent way. The observed lack of glutamate oxidation in brain-specific GDH null CnsGlud1(-/-) mice resulted in a central energy-deprivation state with increased ADP/ATP ratios and phospho-AMPK in the hypothalamus. This induced changes in the autonomous nervous system balance, with increased sympathetic activity promoting hepatic glucose production and mobilization of substrates reshaping peripheral energy stores. Our data reveal the importance of glutamate as necessary energy substrate for the brain and the role of central GDH in the regulation of whole-body energy homeostasis.

KCl -Permeabilized Pancreatic Islets: An Experimental Model to Explore the Messenger Role of ATP in the Mechanism of Insulin Secretion.

Our previous work has demonstrated that islet depolarization with KCl opens connexin36 hemichannels in ß-cells of mouse pancreatic islets allowing the exchange of small metabolites with the extracellular medium. In this study, the opening of these hemichannels has been further characterized in rat islets and INS-1 cells. Taking advantage of hemicannels'opening, the uptake of extracellular ATP and its effect on insulin release were investigated. 70 mM KCl stimulated light emission by luciferin in dispersed rat islets cells transduced with the fire-fly luciferase gene: it was suppressed by 20 mM glucose and 50 µM mefloquine, a specific connexin36 inhibitor. Extracellular ATP was taken up or released by islets depolarized with 70 mM KCl at 5 mM glucose, depending on the external ATP concentration. 1 mM ATP restored the loss of ATP induced by the depolarization itself. ATP concentrations above 5 mM increased islet ATP content and the ATP/ADP ratio. No ATP uptake occurred in non-depolarized or KCl-depolarized islets simultaneously incubated with 50 µM mefloquine or 20 mM glucose. Extracellular ATP potentiated the secretory response induced by 70 mM KCl at 5 mM glucose in perifused rat islets: 5 mM ATP triggered a second phase of insulin release after the initial peak triggered by KCl-depolarization itself; at 10 mM, it increased both the initial, KCl-dependent, peak and stimulated a greater second phase of secretion than at 5 mM. These stimulatory effects of extracellular ATP were almost completely suppressed by 50 µM mefloquine. The magnitude of the second phase of insulin release due to 5 mM extracellular ATP was decreased by addition of 5 mM ADP (extracellular ATP/ADP ratio = 1). ATP acts independently of KATP channels closure and its intracellular concentration and its ATP/ADP ratio seems to regulate the magnitude of both the first (triggering) and second (amplifying) phases of glucose-induced insulin secretion.

Inhibition of connexin 36 hemichannels by glucose contributes to the stimulation of insulin secretion.

The existence of functional connexin36 (Cx36) hemichannels in ß-cells was investigated in pancreatic islets of rat and wild-type (Cx36(+/+)), monoallelic (Cx36(+/-)), and biallelic (Cx36(-/-)) knockout mice. Hemichannel opening by KCl depolarization was studied by measuring ATP release and changes of intracellular ATP (ADP). Cx36(+/+) islets lost ATP after depolarization with 70 mM KCl at 5 mM glucose; ATP loss was prevented by 8 and 20 mM glucose or 50 µM mefloquine (connexin inhibitor). ATP content was higher in Cx36(-/-) than Cx36(+/+) islets and was not decreased by KCl depolarization; Cx36(+/-) islets showed values between that of control and homozygous islets. Five minimolar extracellular ATP increased ATP content and ATP/ADP ratio and induced a biphasic insulin secretion in depolarized Cx36(+/+) and Cx36(+/-) but not Cx36(-/-) islets. Cx36 hemichannels expressed in oocytes opened upon depolarization of membrane potential, and their activation was inhibited by mefloquine and glucose (IC50 ∼8 mM). It is postulated that glucose-induced inhibition of Cx36 hemichannels in islet ß-cells might avoid depolarization-induced ATP loss, allowing an optimum increase of the ATP/ADP ratio by sugar metabolism and a biphasic stimulation of insulin secretion. Gradual suppression of glucose-induced insulin release in Cx36(+/-) and Cx36(-/-) islets confirms that Cx36 gap junction channels are necessary for a full secretory stimulation and might account for the glucose intolerance observed in mice with defective Cx36 expression. Mefloquine targeting of Cx36 on both gap junctions and hemichannels also suppresses glucose-stimulated secretion. By contrast, glucose stimulation of insulin secretion requires Cx36 hemichannels' closure but keeping gap junction channels opened.

Cooperation of taurine uptake and dopamine D1 receptor activation facilitates the induction of protein synthesis-dependent late LTP.

Co-activation of NMDA and dopamine receptors is required for the induction of the late phase of LTP (L-LTP) that is dependent on new protein synthesis. Other neuromodulatory substances may also contribute to this process. Here, we examined whether taurine is one of the neuromodulators contributing to L-LTP induction, since it is known that taurine uptake induces a long-lasting synaptic potentiation dependent on protein synthesis, and taurine uptake inhibition blocks L-LTP induced by tetanization. Experiments were conducted using rat hippocampal slices where field synaptic potentials were evoked and recorded in CA3-CA1 synapses. Taurine (1 mM) applied 10 min before a high frequency stimulation (HFS) train converted a transitory early-LTP (E-LTP) into an L-LTP dependent on protein synthesis. This taurine effect was blocked by a taurine uptake inhibitor. A facilitation of L-LTP induction was also obtained by pre-application of SKF38393, a D1/D5 dopamine receptor (D1R) agonist. In this case, LTP facilitation was not affected by the taurine uptake inhibitor. Nevertheless, when taurine and SKF38393 were simultaneously pre-applied at a concentration that individually did not modify E-LTP, they produced a synergistic mechanism that facilitated the induction of L-LTP with a sole HFS train. This facilitation of L-LTP was blocked by inhibiting either taurine uptake or D1R activation. Taurine and SKF38393 activated different signaling pathways to transform E-LTP into L-LTP. Taurine-induced L-LTP facilitation required MAPK activation, while D1R-agonist-induced facilitation depended mainly on PKA activation and partially on MAPK activation. On the other hand, the synergistic mechanisms induced by the cooperative action of taurine and SKF38393 were impaired by inhibitors against MAPK, PKA and PI3-K. This pharmacological profile resembles that displayed by L-LTP induced by three HFS trains at 10-min intervals. These results indicate that taurine uptake is necessary and cooperates with other neurotransmitter systems in the induction of L-LTP.

HIF2α acts as an mTORC1 activator through the amino acid carrier SLC7A5.

The mammalian target of rapamycin (mTOR) pathway, which is essential for cell proliferation, is repressed in certain cell types in hypoxia. However, hypoxia-inducible factor 2α (HIF2α) can act as a proliferation-promoting factor in some biological settings. This paradoxical situation led us to study whether HIF2α has a specific effect on mTORC1 regulation. Here we show that activation of the HIF2α pathway increases mTORC1 activity by upregulating expression of the amino acid carrier SLC7A5. At the molecular level we also show that HIF2α binds to the Slc7a5 proximal promoter. Our findings identify a link between the oxygen-sensing HIF2α pathway and mTORC1 regulation, revealing the molecular basis of the tumor-promoting properties of HIF2α in von Hippel-Lindau-deficient cells. We also describe relevant physiological scenarios, including those that occur in liver and lung tissue, wherein HIF2α or low-oxygen tension drive mTORC1 activity and SLC7A5 expression.

Delineation of glutamate pathways and secretory responses in pancreatic islets with ß-cell-specific abrogation of the glutamate dehydrogenase.

In pancreatic ß-cells, glutamate dehydrogenase (GDH) modulates insulin secretion, although its function regarding specific secretagogues is unclear. This study investigated the role of GDH using a ß-cell-specific GDH knockout mouse model, called ßGlud1(-/-). The absence of GDH in islets isolated from ßGlud1(-/-) mice resulted in abrogation of insulin release evoked by glutamine combined with 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid or l-leucine. Reintroduction of GDH in ßGlud1(-/-) islets fully restored the secretory response. Regarding glucose stimulation, insulin secretion in islets isolated from ßGlud1(-/-) mice exhibited half of the response measured in control islets. The amplifying pathway, tested at stimulatory glucose concentrations in the presence of KCl and diazoxide, was markedly inhibited in ßGlud1(-/-) islets. On glucose stimulation, net synthesis of glutamate from α-ketoglutarate was impaired in GDH-deficient islets. Accordingly, glucose-induced elevation of glutamate levels observed in control islets was absent in ßGlud1(-/-) islets. Parallel biochemical pathways, namely alanine and aspartate aminotransferases, could not compensate for the lack of GDH. However, the secretory response to glucose was fully restored by the provision of cellular glutamate when ßGlud1(-/-) islets were exposed to dimethyl glutamate. This shows that permissive levels of glutamate are required for the full development of glucose-stimulated insulin secretion and that GDH plays an indispensable role in this process.

Glucose promotion of GABA metabolism contributes to the stimulation of insulin secretion in ß-cells.

We have demonstrated recently that branched-chain α-keto acid stimulation of insulin secretion is dependent on islet GABA (γ-aminobutyric acid) metabolism: GABA transamination to succinic semialdehyde is increased by 2-oxoglutarate, generated in α-keto acid transamination to its corresponding α-amino acid. The present work was aimed at investigating whether glucose also promotes islet GABA metabolism and whether the latter contributes to the stimulation of insulin secretion. Glucose (20 mM) decreased both the content and release of islet GABA. Gabaculine (1 mM), a GABA transaminase inhibitor, partially suppressed the secretory response of rat perifused islets to 20 mM glucose at different L-glutamine concentrations (0, 1 and 10 mM), as well as the glucose-induced decrease in islet GABA. The drug also reduced islet ATP content and the ATP/ADP ratio at 20 mM glucose. Exogenous succinic semialdehyde induced a dose-dependent increase in islet GABA content by reversal of GABA transamination and a biphasic insulin secretion in the absence of glucose. It depolarized isolated ß-cells and triggered action potential firing, accompanied by a reduction of membrane currents through ATP-sensitive K(+) channels. The gene expression and enzyme activity of GABA transaminase were severalfold higher than that of 2-oxoglutarate dehydrogenase in islet homogenates. We conclude that, at high glucose concentrations, there is an increased diversion of glucose metabolism from the citric acid cycle into the 'GABA shunt'. Semialdehyde succinic acid is a cell-permeant 'GABA-shunt' metabolite that increases ATP and the ATP/ADP ratio, depolarizes ß-cells and stimulates insulin secretion. In summary, an increased islet GABA metabolism may trigger insulin secretion.

Branched-chain 2-oxoacid transamination increases GABA-shunt metabolism and insulin secretion in isolated islets.

We have previously shown that oxo-4-methylpentanoate promotes islet GABA (gamma-aminobutyric acid) metabolism and stimulates insulin secretion. The main aim of this work was to explore the participation of the transamination of branched-chain 2-oxoacids in these processes with the aid of several inhibitors of this enzyme activity. No correlation was found between the transamination of branched-chain 2-oxoacids in islet homogenates and insulin secretion. However, in vivo transamination rates correlated better with the secretion capacity of the different branched-chain 2-oxoacids. Gabapentin, a specific inhibitor of the cytosolic isoenzyme, showed greater potential to decrease the in vitro transamination rates of oxo-3-methylbutyrate and oxo-3-methylpentanoate than those of oxo-4-methylpentanoate and oxohexanoate; this correlated with its capacity to decrease insulin secretion. 4-Methylvaleric acid very strongly inhibited the transamination of all the branched-chain 2-oxoacids and blocked their capacity to decrease islet GABA and to stimulate insulin secretion. KCl at 70 mM at stimulated islet GABA release, subsequently decreasing its tissue concentration. This 'non-metabolic' decrease of GABA suppressed the second phase of insulin secretion triggered by oxo-4-methylpentanoate and oxohexanoate. Oxo-4-methylpentanoate and oxo-3-methylpentanoate suppressed dose-dependent 2-oxoglutarate dehydrogenase activity in islet homogenates. In conclusion, the transamination of branchedchain 2-oxoacids is more important to the stimulation of insulin secretion than their catabolism, and transamination decreases islet GABA concentrations by promoting GABA metabolism. Also, inhibition of 2-oxoglutarate dehydrogenase by branched-chain 2-oxoacids may increase metabolic flux in the 'GABA-shunt' at the expense of reduced tricarboxylic acid cycle flux.

Oxo-4-methylpentanoic acid directs the metabolism of GABA into the Krebs cycle in rat pancreatic islets.

OMP (oxo-4-methylpentanoic acid) stimulates by itself a biphasic secretion of insulin whereas L-leucine requires the presence of L-glutamine. L-Glutamine is predominantly converted into GABA (gamma-aminobutyric acid) in rat islets and L-leucine seems to promote its metabolism in the 'GABA shunt' [Fernández-Pascual, Mukala-Nsengu-Tshibangu, Martín del Río and Tamarit-Rodríguez (2004) Biochem. J. 379, 721-729]. In the present study, we have investigated how 10 mM OMP affects L-glutamine metabolism to uncover possible differences with L-leucine that might help to elucidate whether they share a common mechanism of stimulation of insulin secretion. In contrast with L-leucine, OMP alone stimulated a biphasic insulin secretion in rat perifused islets and decreased the islet content of GABA without modifying its extracellular release irrespective of the concentration of L-glutamine in the medium. GABA was transaminated to L-leucine whose intracellular concentration did not change because it was efficiently transported out of the islet cells. The L-[U-14C]-Glutamine (at 0.5 and 10.0 mM) conversion to 14CO2 was enhanced by 10 mM OMP within 30% and 70% respectively. Gabaculine (250 microM), a GABA transaminase inhibitor, suppressed OMP-induced oxygen consumption but not L-leucine- or glucose-stimulated respiration. It also suppressed the OMP-induced decrease in islet GABA content and the OMP-induced increase in insulin release. These results support the view that OMP promotes islet metabolism in the 'GABA shunt' generating 2-oxo-glutarate, in the branched-chain alpha-amino acid transaminase reaction, which would in turn trigger GABA deamination by GABA transaminase. OMP, but not L-leucine, suppressed islet semialdehyde succinic acid reductase activity and this might shift the metabolic flux of the 'GABA shunt' from gamma-hydroxybutyrate to succinic acid production.

Granulocyte-macrophage colony-stimulating factor increases L-arginine transport through the induction of CAT2 in bone marrow-derived macrophages.

L-arginine transport is crucial for macrophage activation because it supplies substrate for the key enzymes nitric oxide synthase 2 and arginase I. These enzymes participate in classic and alternative activation of macrophages, respectively. Classic activation of macrophages is induced by type I cytokines, and alternative activation is induced by type II cytokines. The granulocyte macrophage colony-stimulating factor (GM-CSF), in addition to inducing proliferation and differentiation of macrophages, activates arginase I, but its action on L-arginine transport is unknown. We studied the L-arginine transporters that are active in mouse primary bone marrow-derived macrophages (BMM) and examined the effect of GM-CSF treatment on transport activities. Under basal conditions, L-arginine entered mainly through system y(+)L (>75%). The remaining transport was explained by system y(+) (<10%) and a diffusion component (10-15%). In response to GM-CSF treatment, transport activity increased mostly through system y(+) (>10-fold), accounting for about 40% of the total L-arginine transport. The increase in y(+) activity correlated with a rise in cationic amino acid transporter (CAT)-2 mRNA and protein. Furthermore, GM-CSF induced an increase in arginase activity and in the conversion of L-arginine to ornithine, citrulline, glutamate, proline, and polyamines. BMM obtained from CAT2-knockout mice responded to GM-CSF by increasing arginase activity and the expression of CAT1 mRNA, which also encodes system y(+) activity. Nonetheless, the increase in CAT1 activity only partially compensated the lack of CAT2 and L-arginine metabolism was hardly stimulated. We conclude that BMM present mainly y(+)L activity and that, in response to GM-CSF, l-arginine transport augments through CAT2, thereby increasing the availability of this amino acid to the cell.

Protective effect of L-trans-pyrrolidine-2,4-dicarboxilic acid preload against cell death induced by oxygen/glucose deprivation in differentiated PC12 cells.

It has been postulated that cellular glutamate is released into the extracellular fluid when the energy supply of the brain is compromised (i.e., anoxia or oxygen/glucose deprivation), and there the amino acid triggers the so-called excitotoxic cascade, causing neuronal death. Several mechanisms for this release have been postulated, and, by using glutamate transporter inhibitors, several authors have established that reversed uptake is the major mechanism through which glutamate is released in acute oxygen/glucose deprivation. We have studied the effect of the slowly transported glutamate analogue L-trans-pyrrolidine-2,4-dicarboxilic acid (PDC) preload on glutamate release and cell death in an in vitro model of oxygen plus glucose deprivation with differentiated PC12 cells. As expected, we found that PDC preload inhibits glutamate release induced by oxygen/glucose deprivation, supporting the conclusion that it occurs via reverse transport. In addition, we show that PDC preload but not the nontransportable glutamate uptake inhibitor DL-threo-beta-benzyloxyaspartate (TBOA) protects cells against the death induced by oxygen/glucose deprivation, indicating that PDC entry into the cell is necessary for this protective effect. This protection does not correlate with the extracellular glutamate concentration or changes in proteins synthesis rate and eukaryotic initiation 2 phosphorylation. Oxygen/glucose deprivation induces a significant increase in glutathione levels in both unloaded and PDC-preloaded cells, but this increase is not due to up-regulation of glutamate cysteine ligase levels. Intracellular glutathione disulfide (GSSG) significantly increased after oxygen/glucose deprivation. It was also interesting that intracellular GSSG levels in PDC-preloaded cells under oxygen/glucose deprivation strongly correlate with the protection exerted by this compound against cell death.

Similar effects of succinic acid dimethyl ester and glucose on islet calcium oscillations and insulin release.

UNLABELLED: The relative contribution of glycolysis vs. oxidative metabolism to the stimulus secretion coupling mechanism of beta-cells was investigated in isolated islets. For that purpose, the secretory and intracellular calcium responses of islets to both glucose and succinic acid dimethyl ester (SAD) were compared. After 45 min of rat islet perifusion in the absence of substrates, the maximum secretory responses to glucose (20 mmol/L) and SAD (10 mmol/L) were qualitatively and quantitatively indistinguishable. Malonic acid dimethyl ester (a permeable citric acid cycle inhibitor) suppressed the insulin secretory response to both 20 mmol/L glucose and 10 mmol/L SAD (-70% on average). The inhibitor decreased within 70% the rate of 14CO2-production from 10 mmol/L [2-(14)C]pyruvate without affecting the rate of 20 mmol/L D-[5-(3)H]glucose utilization. Both, 11.1 mmol/L glucose and 10 mmol/L SAD, elevated the intracellular calcium concentration and induced a similar pattern of oscillations that were rapidly ablated by 20 mmol/L malonic acid dimethyl ester. However, the intracellular concentration of calcium declined to basal values several minutes after the introduction of the inhibitor in the presence of SAD whereas it remained elevated in the case of glucose. IN CONCLUSION: (1) An exclusive increase of mitochondrial metabolism in pancreatic islets was sufficient to mimic the effects of glucose on intracellular calcium and insulin secretion. (2) Islet glycolysis and/or the re-oxidation of cytoplasmic NADH allowed the maintenance of an elevated, though non-oscillating, intracellular calcium concentration, but a reduced response to glucose.

Role of taurine uptake on the induction of long-term synaptic potentiation.

Taurine application in the CA1 area of rat hippocampal slices induces a long-lasting potentiation of excitatory synaptic transmission that has some mechanistic similitude with the late phase of long-term potentiation (L-LTP). Previous indirect evidence such as temperature and sodium dependence indicated that taurine uptake is one of the primary steps leading to the taurine-induced synaptic potentiation. We show that taurine-induced potentiation is not related to the intracellular accumulation of taurine and is not impaired by 2-guanidinoethanesulphonic acid, a taurine transport inhibitor that is a substrate of taurine transporter. We have found that taurine uptake in hippocampal synaptosomes was inhibited by SKF 89976A, a GABA uptake blocker that is not transportable by GABA transporters. SKF 89976A prevents the induction of synaptic potentiation by taurine application. This effect is neither mimicked by nipecotic acid, a broad inhibitor of GABA transporters that does not affect taurine uptake, nor by NO-711, a specific and potent inhibitor of GABA transporter GAT-1. In addition, L-LTP induced by trains of high-frequency stimulation is also inhibited by SKF 89976A, and taurine, at a concentration that does not change basal synaptic transmission, overcomes such inhibition. We conclude that taurine induces synaptic potentiation through the activation of a system transporting taurine and that taurine uptake is required for the induction of synaptic plasticity phenomena such as L-LTP.

Conversion into GABA (gamma-aminobutyric acid) may reduce the capacity of L-glutamine as an insulin secretagogue.

We have carried out a detailed examination of L-glutamine metabolism in rat islets in order to elucidate the paradoxical failure of L-glutamine to stimulate insulin secretion. L-Glutamine was converted by isolated islets into GABA (gamma-aminobutyric acid), L-aspartate and L-glutamate. Saturation of the intracellular concentrations of all of these amino acids occurred at approx. 10 mmol/l L-glutamine, and their half-maximal values were attained at progressively increasing concentrations of L-glutamine (0.3 mmol/l for GABA; 0.5 and 1.0 mmol/l for Asp and Glu respectively). GABA accumulation accounted for most of the 14CO2 produced at various L-[U-14C]glutamine concentrations. Potentiation by L-glutamine of L-leucine-induced insulin secretion in perifused islets was suppressed by malonic acid dimethyl ester, was accompanied by a significant decrease in islet GABA accumulation, and was not modified in the presence of GABA receptor antagonists [50 micromol/l saclofen or 10 micromol/l (+)-bicuculline]. L-Leucine activated islet glutamate dehydrogenase activity, but had no effect on either glutamate decarboxylase or GABA transaminase activity, in islet homogenates. We conclude that (i) L-glutamine is metabolized preferentially to GABA and L-aspartate, which accumulate in islets, thus preventing its complete oxidation in the Krebs cycle, which accounts for its failure to stimulate insulin secretion; (ii) potentiation by L-glutamine of L-leucine-induced insulin secretion involves increased metabolism of L-glutamate and GABA via the Krebs cycle (glutamate dehydrogenase activation) and the GABA shunt (2-oxoglutarate availability for GABA transaminase) respectively, and (iii) islet release of GABA does not seem to play an important role in the modulation of the islet secretory response to the combination of L-leucine and L-glutamine.

Cystinuria-specific rBAT(R365W) mutation reveals two translocation pathways in the amino acid transporter rBAT-b0,+AT.

Apical reabsorption of dibasic amino acids and cystine in kidney is mediated by the heteromeric amino acid antiporter rBAT/b(0,+)AT (system b(0,+)). Mutations in rBAT cause cystinuria type A, whereas mutations in b(0,+)AT cause cystinuria type B. b(0,+)AT is the catalytic subunit, whereas it is believed that rBAT helps the routing of the rBAT/b(0,+)AT heterodimeric complex to the plasma membrane. In the present study, we have functionally characterized the cystinuria-specific R365W (Arg(365)-->Trp) mutation of human rBAT, which in addition to a trafficking defect, alters functional properties of the b(0,+) transporter. In oocytes, where human rBAT interacts with the endogenous b(0,+)AT subunit to form an active transporter, the rBAT(R365W) mutation caused a defect of arginine efflux without altering arginine influx or apparent affinities for intracellular or extracellular arginine. Transport of lysine or leucine remained unaffected. In HeLa cells, functional expression of rBAT(R365W)/b(0,+)AT was observed only at the permissive temperature of 33 degrees C. Under these conditions, the mutated transporter showed 50% reduction of arginine influx and a similar decreased accumulation of dibasic amino acids. Efflux of arginine through the rBAT(R365W)/b(0,+)AT holotransporter was completely abolished. This supports a two-translocation-pathway model for antiporter b(0,+), in which the efflux pathway in the rBAT(R365W)/b(0,+)AT holotransporter is defective for arginine translocation or dissociation. This is the first direct evidence that mutations in rBAT may modify transport properties of system b(0,+).

Basolateral LAT-2 has a major role in the transepithelial flux of L-cystine in the renal proximal tubule cell line OK.

During renal reabsorption, the amino acid transporters b(o,+) and y(+)L have a major role in the apical uptake of cystine and dibasic amino acids and in the basolateral efflux of dibasic amino acids, respectively. In contrast, the transporters responsible for the basolateral efflux of the apically transported cystine are unknown. This study shows the expression of system L and y(+)L transport activities in the basolateral domain of the proximal tubule-derived cell line OK and the cloning of the corresponding LAT-2 and y(+)LAT-1 cDNAs. Stable transfection with a LAT-2 antisense sequence demonstrated the specific role of LAT-2 in the basolateral system L amino acid exchange activity in OK cells. This partial reduction of LAT-2 expression decreased apical-to-basolateral trans-epithelial flux of cystine and resulted in a twofold to threefold increase in the intracellular content of cysteine. In contrast, the content of serine, threonine, and alanine showed a tendency to decrease, whereas other LAT-2 substrates were not affected. This demonstrates that LAT-2 plays a major specific role in the net basolateral efflux of cysteine and points to LAT-2 as a candidate gene to modulate cystine reabsorption.