Ceftriaxone regulates glutamate production and vesicular assembly in presynaptic terminals through GLT-1 in APP/PS1 mice.

Perturbations in the glutamate-glutamine cycle and glutamate release from presynaptic terminals have been involved in the development of cognitive deficits in Alzheimer's disease (AD) patients and mouse models. Glutamate transporter-1 (GLT-1) removes glutamate from the synaptic cleft and transports it into astrocytes, where it is used as substrate for the glutamate-glutamine cycle. Ceftriaxone has been reported to improve cognitive deficits in AD mice by increasing GLT-1 expression, glutamate transformation to glutamine, and glutamine efflux from astrocytes. However, the impact of ceftriaxone on glutamine metabolism in neurons is unknown. The present study aimed to investigate whether ceftriaxone regulated the production and vesicular assembly of glutamate in the presynaptic terminals of neurons and to determine GLT-1 involvement in this process. We used the amyloid precursor protein (APP)/presenilin-1 (PS1) AD mouse model and GLT-1 knockdown APP/PS1 (GLT-1+/-/APP/PS1) mice. The expression levels of sodium-coupled neutral amino-acid transporter 1 (SNAT1) and vesicular glutamate transporters 1 and 2 (VGLUT1/2) were analyzed by immunofluorescence and immunohistochemistry staining as well as by Western blotting. Glutaminase activity was assayed by fluorometry. Ceftriaxone treatment significantly increased SNAT1 expression and glutaminase activity in neurons in APP/PS1 mice. Similarly, VGLUT1/2 levels were increased in the presynaptic terminals of APP/PS1 mice treated with ceftriaxone. The deletion of one GLT-1 allele in APP/PS1 mice prevented the ceftriaxone-induced upregulation of SNAT1 and VGLUT1/2 expression, indicating that GLT-1 played an important role in ceftriaxone effect. Based on the role of SNAT1, glutaminase, and VGLUT1/2 in the glutamate-glutamine cycle in neurons, the present results suggested that ceftriaxone improved the production and vesicular assembly of glutamate as a neurotransmitter in presynaptic terminals by acting on GLT-1 in APP/PS1 mice.

Pulsatile delivery of a leucine supplement during long-term continuous enteral feeding enhances lean growth in term neonatal pigs.

Neonatal pigs are used as a model to study and optimize the clinical treatment of infants who are unable to maintain oral feeding. Using this model, we have shown previously that pulsatile administration of leucine during continuous feeding over 24 h via orogastric tube enhanced protein synthesis in skeletal muscle compared with continuous feeding alone. To determine the long-term effects of leucine pulses, neonatal piglets (n = 11-12/group) were continuously fed formula via orogastric tube for 21 days, with an additional parenteral infusion of either leucine (CON + LEU; 800 µmol·kg-1·h-1) or alanine (CON + ALA) for 1 h every 4 h. The results show that body and muscle weights and lean gain were ∼25% greater, and fat gain was 48% lower in CON + LEU than CON + ALA; weights of other tissues were unaffected by treatment. Fractional protein synthesis rates in longissimus dorsi, gastrocnemius, and soleus muscles were ∼30% higher in CON + LEU compared with CON + ALA and were associated with decreased Deptor abundance and increased mTORC1, mTORC2, 4E-BP1, and S6K1 phosphorylation, SNAT2 abundance, and association of eIF4E with eIF4G and RagC with mTOR. There were no treatment effects on PKB, eIF2α, eEF2, or PRAS40 phosphorylation, Rheb, SLC38A9, v-ATPase, LAMTOR1, LAMTOR2, RagA, RagC, and LAT1 abundance, the proportion of polysomes to nonpolysomes, or the proportion of mRNAs encoding rpS4 or rpS8 associated with polysomes. Our results demonstrate that pulsatile delivery of a leucine supplement during 21 days of continuous enteral feeding enhances lean growth by stimulating the mTORC1-dependent translation initiation pathway, leading to protein synthesis in skeletal muscle of neonates.

Cortisol stimulates system A amino acid transport and SNAT2 expression in a human placental cell line (BeWo).

Both placental system A activity and fetal plasma cortisol concentrations are associated with intrauterine growth retardation, but it is not known if these factors are mechanistically related. Previous functional studies using hepatoma cells and fibroblasts produced conflicting results regarding the regulation of system A by cortisol. Using the b30 BeWo choriocarcinoma cell line, we investigated the regulation of system A by cortisol. System A function was analyzed using methyl amino isobutyric acid (MeAIB) transcellular transport studies. Transporter expression [system A transporter (SNAT)1/2] was studied at the mRNA and protein levels using Northern and Western blotting, respectively. Localization was carried out using immunocytochemistry. The [(14)C]MeAIB transfer rate across BeWo monolayers after preincubation with cortisol for 24 h was significantly increased compared with control. This was associated with a relocalization of the SNAT2 transporter at lower cortisol levels and significant upregulation of mRNA and protein expression levels at cortisol levels >1 microM. This is the first study to show functional and molecular regulation of system A by cortisol in BeWo cells. It is also the first study to identify which system A isoform is regulated. These results suggest that cortisol may be involved in upregulation of system A in the placenta to ensure sufficient amino acid supply to the developing fetus.

Modulation of amino acid uptake by TGF-beta in lung myofibroblasts.

Hormones such as insulin, growth factors, and cell stress stimulate system A amino acid transporter. Transforming growth factor-beta (TGF-beta) stimulates amino acid uptake thereby inducing cell proliferation, cellular hypertrophy, and matrix synthesis. Insulin appears to activate amino acid in smooth muscle cells via a phosphatidylinositol 3-kinase (PI3-kinase)-dependent pathway. We examine the effect and interaction of TGF-beta, insulin, and PI3-kinase activity on amino acid uptake in human lung myofibroblasts. TGF-beta treatment induced large increases in system A activity and a small delayed increase in the phosphorylation of protein kinase B, also termed phospho-Akt. In contrast, insulin induced small increases in system A activity and large increases in phospho-Akt levels. LY294002, a PI3-kinase inhibitor, blocked the TGF-beta-induced amino acid uptake only partially, but completely blocked TGF-beta-induced Akt phosphorylation. Moreover, the level of phospho-Smad3 was found to be high even when LY294002 blocked TGF-beta-induced phospho-Akt levels. Inhibition of PI3-kinase activity resulted in increase in Km, consistent with a major change in transporter activity without change in transporter number. The PI3-kinase inhibitor also did not change the amino acid transporter 2 (ATA2) mRNA levels. Taken together, these results suggest that TGF-beta induced Smad-3 and amino acid uptake through a PI3-kinase independent pathway.

Impact of forskolin and amino acid depletion upon System A activity and SNAT expression in BeWo cells.

Amino acid transport System A (SysA) plays an important role in mediating the transplacental transfer of neutral amino acids from mother to fetus. Given that prior work has demonstrated that SysA activity is regulated, both over gestation and in response to dietary restriction during pregnancy, we examined the response of SysA activity and sodium-dependent neutral amino acid transporter (SNAT; responsible for SysA activity) expression to cAMP analogues and amino acid deprivation in BeWo cells, an accepted model of placental syncytia. SysA activity was unaffected by forskolin, a cAMP agonist, at 48 and 72 h. Amino acid depletion was associated with an up-regulation of SysA activity, largely mediated through an enhancement of SNAT2 (Slc38a2) expression at both the protein and mRNA level. SNAT1 (Slc38a1) expression did not change in response to amino acid depletion, while SNAT4 (Slc38a4) could not be detected. In summary, SysA activity in BeWo cells responds to amino acid depletion through the differential regulation of SNAT subtypes.

Ceramide down-regulates System A amino acid transport and protein synthesis in rat skeletal muscle cells.

Skeletal muscle is a major insulin target tissue and has a prominent role in the control of body amino acid economy, being the principal store of free and protein-bound amino acids and a dominant locus for amino acid metabolism. Interplay between diverse stimuli (e.g., hormonal/nutritional/mechanical) modulates muscle insulin action to serve physiological need through the action of factors such as intramuscular signaling molecules. Ceramide, a product of sphingolipid metabolism and cytokine signaling, has a potent contra-insulin action with respect to the transport and deposition of glucose in skeletal muscle, although ceramide effects on muscle amino acid turnover have not previously been documented. Here, membrane permeant C2-ceramide is shown to attenuate the basal and insulin-stimulated activity of the Na+-dependent System A amino acid transporter in rat muscle cells (L6 myotubes) by depletion of the plasma membrane abundance of SNAT2 (a System A isoform). Concomitant with transporter down-regulation, ceramide diminished both intramyocellular amino acid abundance and the phosphorylation of translation regulators lying downstream of mTOR. The physiological outcome of ceramide signaling in this instance is a marked reduction in cellular protein synthesis, a result that is likely to represent an important component of the processes leading to muscle wasting in catabolic conditions.

The synthesis of SNAT2 transporters is required for the hypertonic stimulation of system A transport activity.

In cultured human fibroblasts incubated under hypertonic conditions, the stimulation of system A for neutral amino acid transport, associated to the increased expression of the mRNA for SNAT2 transporter, leads to an expanded intracellular amino acid pool and to the recovery of cell volume. A protein of nearly 60 kDa, recognized by an antiserum against SNAT2, is increased both in the pool of biotinylated membrane proteins and in the total cell lysate of hypertonically stressed cells. The increased level of SNAT2 transporters in hypertonically stressed cells is confirmed by immunocytochemistry. DRB, an inhibitor of transcription, substantially inhibits the increase of SNAT2 proteins on the plasma membrane, completely suppresses the stimulation of system A transport activity, and markedly delays the cell volume recovery observed during the hypertonic treatment. On the contrary, if the transport activity of system A is adaptively increased by amino acid starvation in the presence of DRB, the increase of SNAT2 transporters on the plasma membrane is still clearly detectable and the transport change only partially inhibited. It is concluded that the synthesis of new SNAT2 transporters is essential for the hypertonic stimulation of transport system A, but accounts only in part for the adaptive increase of the system.

Growth hormone and epidermal growth factor together enhance amino acid transport systems B0,+ and A in remnant small intestine after massive enterectomy.

BACKGROUND: Sodium-dependent brush-border nutrient transport is decreased 2 weeks after massive enterectomy. This down-regulation is ameliorated by a 1-week infusion of parenteral growth hormone (GH) and epidermal growth factor (EGF) started 1 week after resection. We hypothesize that glutamine (GLN) transport will be enhanced by earlier and longer growth factor infusion, with differential effects on the Na(+)-dependent GLN transport systems A, B(0,+), and B(0)/ASCT2. MATERIALS AND METHODS: New Zealand White rabbits underwent 70% small bowel resection then immediately received parenteral EGF, GH, both EGF and GH, or neither for 2 weeks. Na(+)-dependent 3H-GLN uptake by jejunal and ileal brush-border membrane vesicles was measured and the contribution of systems A, B(0,+), and B(0) was then determined by competitive inhibition. Data were analyzed using one-way analysis of variance. RESULTS: In nonresected animals, the relative contribution of the systems was similar in jejunum (A 9%, B(0,+) 20%, and B(0) 71%) and ileum (A 13%, B(0,+) 27%, and B(0) 60%). Na(+)-dependent GLN uptake was reduced by one half in resected untreated controls, primarily because of decreased B(0) activity. EGF or GH alone did not affect Na(+)-dependent GLN transport, but, as a combination, there was increased uptake in the residual ileum and jejunum by 144% and 150%, respectively, over resected controls (P < 0.05). This was twice that achieved by delayed and shorter-duration combination treatment. This augmentation was a result of a 6.1-8.2-fold increase in system A as well as a 3.8-3.9-fold enhancement of system B(0,+) activity in remnant ileum and jejunum (P < 0.01). CONCLUSIONS: Parenteral EGF and GH, given in combination for 2 weeks immediately after massive enterectomy, synergistically enhance GLN uptake by systems A and B(0,+).

Regulation of L-alanine transport systems A and ASC by cyclic AMP and calcium in a reptilian duodenal model.

The regulation of neutral amino acid transport by cyclic AMP (cAMP) and calcium across the isolated duodenum of the lizard Gallotia galloti has been studied under short-circuit conditions. Active L-alanine transport was stimulated by forskolin, theophylline and dibutyryl cyclic AMP (db-cAMP). All these agents increased transmural potential difference (PD) and short-circuit current (I(sc)) in a manner consistent with the activation of a chloride secretory pathway. Both forskolin and theophylline increased intracellular cAMP levels in the lizard duodenal mucosa. Addition of calcium ionophore A23187 rapidly reduced mucosa-to-serosa L-alanine fluxes and diminished net L-alanine transport. Despite the reduction of alanine fluxes by A23187, transepithelial PD and I(sc) values were increased by the ionophore. Analyses of the responses of isolated transport pathways indicated that the Na(+)-independent L-alanine transport system was unaffected by db-cAMP or calcium ionophore. By contrast, Na(+)-dependent transport activities were profoundly modified by these agents. Thus, while system A [alpha-methylamino-isobutiric acid (MeAIB)-transporting pathway] was stimulated by increased calcium, system ASC activity was nearly abolished. Calcium ionophore also potentiated the electrogenic response of system A. Forskolin strongly stimulated system ASC activity but left system A activity unchanged. Activation of system ASC by forskolin was clearly electroneutral, as pre-incubation of the tissues with the chloride channel blocker diphenylamine-2-carboxilic acid (DPC) completely prevented forskolin-induced transepithelial electrical responses. It is concluded that intracellular messengers cAMP and calcium oppositely modulate active Na(+)-dependent (L)-alanine transport in the lizard intestine. The different sensitivity exhibited by individual transport pathways may well account for the changes observed in overall alanine transport.

Leptin stimulates the activity of the system A amino acid transporter in human placental villous fragments.

The activity and expression of placental nutrient transporters are primary determinants for the supply of nutrients to the fetus, and these nutrients in turn regulate fetal growth. We developed an experimental system to assess amino acid uptake in single primary villous fragments to study hormonal regulation of the amino acid transporter system A in term human placenta. Validation of the method, using electron microscopy and studies of hormone production, indicated that fragments maintained ultrastructural and functional integrity for at least 3 h. The activity of system A was measured as the Na(+)-dependent uptake of methylaminoisobutyric acid (MeAIB), and the effect of 1 h incubation in various hormones was investigated. Uptake of MeAIB into villous fragments in the presence of Na(+) was linear up to at least 30 min. Insulin (300 ng/ml, n = 14) increased system A activity by 56% (P < 0.05). This effect was also present at insulin concentrations in the physiological range (+47% at 0.6 ng/ml, n = 10, P < 0.05). Leptin (500 ng/ml, n = 14) increased Na(+)-dependent MeAIB uptake by 37% (P < 0.05). System A activity increased in a concentration-dependent fashion in response to leptin (n = 10). However, neither epidermal GF (600 ng/ml), cortisol (340 ng/ml), nor GH (500 ng/ml) altered system A activity significantly (n = 14). We conclude that primary single isolated villous fragments can be used in studies of hormonal regulation of nutrient uptake into the syncytiotrophoblast. These data suggest that leptin regulates system A, a key amino acid transporter.

Inhibition of system A amino acid transport activity by ethanol in BeWo choriocarcinoma cells.

OBJECTIVE: Our purpose was to investigate the influence of ethanol on system A amino acid transporter in BeWo cells. STUDY DESIGN: BeWo cells were cultured in the absence or presence of ethanol. The function of system A was monitored by the transport of alpha-(methylamino)isobutyric acid. Messenger RNA levels for system A were assessed by Northern analysis. RESULTS: Treatment of BeWo cells with ethanol reduced the activity of system A. The effect was dose and treatment time dependent. The decrease in system A activity was 38% +/- 3% at 0.75% ethanol with a 16-hour treatment time. The activities of several other transporters tested were not affected. The effect on system A activity was associated with a decrease in the maximal velocity of the transport system without affecting the substrate affinity. Ethanol did not alter the messenger RNA levels for system A. CONCLUSION: Exposure of BeWo cells to ethanol significantly reduces the function of system A. This finding has potential implications that may be relevant to the pathogenesis of the fetal alcohol syndrome.