BCAAs and Di-Alanine supplementation in the prevention of skeletal muscle atrophy: preclinical evaluation in a murine model of hind limb unloading.

Skeletal muscle atrophy occurs in response to various pathophysiological stimuli, including disuse, aging, and neuromuscular disorders, mainly due to an imbalance of anabolic/catabolic signaling. Branched Chain Amino Acids (BCAAs: leucine, isoleucine, valine) supplements can be beneficial for counteracting muscle atrophy, in virtue of their reported anabolic properties. Here, we carried out a proof-of-concept study to assess the in vivo/ex vivo effects of a 4-week treatment with BCAAs on disuse-induced atrophy, in a murine model of hind limb unloading (HU). BCAAs were formulated in drinking water, alone, or plus two equivalents of L-Alanine (2 ALA) or the dipeptide L-Alanyl-L-Alanine (Di-ALA), to boost BCAAs bioavailability. HU mice were characterized by reduction of body mass, decrease of soleus - SOL - muscle mass and total protein, alteration of postural muscles architecture and fiber size, dysregulation of atrophy-related genes (Atrogin-1, MuRF-1, mTOR, Mstn). In parallel, we provided new robust readouts in the HU murine model, such as impaired in vivo isometric torque and ex vivo SOL muscle contractility and elasticity, as well as altered immune response. An acute pharmacokinetic study confirmed that L-ALA, also as dipeptide, enhanced plasma exposure of BCAAs. Globally, the most sensitive parameters to BCAAs action were muscle atrophy and myofiber cross-sectional area, muscle force and compliance to stress, protein synthesis via mTOR and innate immunity, with the new BCAAs + Di-ALA formulation being the most effective treatment. Our results support the working hypothesis and highlight the importance of developing innovative formulations to optimize BCAAs biodistribution.

Lipid Nanoemulsions of Rebamipide: Formulation, Characterization, and In Vivo Evaluation of Pharmacokinetic and Pharmacodynamic Effects.

Rebamipide has low oral bioavailability (10%) due to its low solubility and permeability. Lipid nanoemulsions (LNEs) were prepared in order to improve its oral bioavailability. Rebamipide-loaded lipid nanoemulsions were formulated by hot homogenization and ultrasonication method. Olive oil and egg lecithin in various concentrations as emulsifier were used in the preparation of LNEs. The lipid nanoemulsions were evaluated for various parameters. The globule size, polydispersity index (PDI), and zeta potential (ZP) of the formulations ranged from 230.3 ± 3.88 to 279.8 ± 5.76 nm, 0.204 ± 0.008 to 0.246 ± 0.029, and - 27.7 ± 2.05 to - 31.0 ± 1.87 mV, respectively. Entrapment efficiency and assay values ranged from 99.90 ± 0.006 to 99.92 ± 0.002% and 99.3 ± 0.808 to 99.6 ± 0.360, respectively. Physical stability test results revealed that the optimized LNEs were stable for 2 months at both room (25°C) and refrigerated temperature (4°C). The optimized LNE showed 4.32-fold improvement in the oral bioavailability in comparison to a marketed tablet suspension. In vivo anti ulcer activity of rebamipide LNE was studied by testing the prophylactic effect in preventing the mucosal damage in stomach region. The mucosa of stomach in animals was damaged by per oral administration of 80% alcohol. Maximum prophylactic antiulcer activity was observed by per oral delivery of rebamipide as LNE. Our results indicated that LNEs were a promising approach for the oral delivery of rebamipide for systemic effects along with local effects in protecting gastric region, which gets damaged during peptic ulcers.

Pharmacokinetic drug evaluation of safinamide mesylate for the treatment of mid-to-late stage Parkinson's disease.

INTRODUCTION: Patients with Parkinson's disease suffer from a heterogeneous expression of neurotransmitter deficits. They cause an individual variable expression of motor and non-motor symptoms. Thus, drugs with various mechanisms of actions are suitable to counteract these disease related neurotransmitter alterations. Areas covered: This invited review suggests safinamide as an ideal compound for therapy of Parkinson's disease, as its pharmacological profile includes reversible monoamine oxidase B inhibition, blockage of voltage-dependent sodium channels, modulation of calcium channels and abnormal glutamate release. Safinamide may provide benefits effects on non-motor symptoms in addition to the demonstrated amelioration of motor impairment in levodopa treated patients with Parkinson's disease. Safinamide was well tolerated and safe when administered in dose of 50 or 100 mg daily in pivotal trials. Expert opinion: Clinical handling, safety and tolerability of Safinamide are better than of dopamine agonists or levodopa. Safinamide supplements the existing armamentarium of drugs for Parkinson's disease. Safinamide will help to reduce dosing of levodopa but also of dopamine agonists during long term treatment in patients with Parkinson's disease.

Determining the functional role of waterborne amino acid uptake in hagfish nutrition: a constitutive pathway when fasting or a supplementary pathway when feeding?

Hagfish are unique among aquatic "vertebrates" in their ability to absorb amino acids directly from the water via skin and gill epithelia, but it is unknown whether this phenomenon extends beyond a few studied substrates; what effect fed state has on absorption; and what functional role this may play in hagfish nutrition. Using in vivo and in vitro transport assays, uptake and tissue distribution of the waterborne amino acids L-alanine, L-lysine, and L-phenylalanine were examined as a function of fed state. All three amino acids were shown to be taken up from the water (lysine and phenylalanine for the first time). Following immersion in radiolabelled solutions for 24 h, phenylalanine was the amino acid that accumulated at the highest levels in almost all tissues, with the highest accumulation noted in red blood cells and bile, followed by gill and liver. In general, tissues of fed hagfish displayed a significantly reduced phenylalanine accumulation compared to tissues of hagfish fasted for 3 weeks. An in vitro assay showed that phenylalanine was transported across the skin at the highest rate, with the uptake of lysine occurring at the lowest rate. Feeding status had no significant effect on in vitro transport. These data indicate that dissolved organic nutrients are a significant source of nutrition to hagfish, and may be relatively more important during periods of fasting than during periods of feeding when immersed in decaying carcasses.

Adjuvant therapies for Parkinson's disease: critical evaluation of safinamide.

Safinamide (SAF) is a new drug developed for the treatment of Parkinson's disease (PD). It is a benzylamino derivative with multiple mechanisms of action and antiparkinsonian, anticonvulsant, and neuroprotective properties. SAF inhibits monoamine oxidase B and dopamine reuptake and glutamate release, blocks voltage-dependent sodium channels, and modulates calcium channels. Although the antiparkinsonian effect can be ascribed in part to the inhibition of the monoamine oxidase B, which is complete at 50 mg, the enhanced benefit seen at the 100 mg dose is probably due to nondopaminergic mechanisms. SAF will represent an important option for patients with both early and advanced PD. In early PD patients, the addition of SAF to dopamine agonists may be an effective treatment strategy to improve motor function, prolong the use of dopamine agonists, and/or delay the introduction of levodopa. In advanced parkinsonian patients, SAF has been demonstrated to significantly increase on time with no, or nontroublesome dyskinesias. All studies performed have demonstrated its efficacy in benefiting both short-term and long-term quality-of-life outcomes in both early and advanced PD patients. SAF has been investigated in long-term (24 months), double-blind, placebo-controlled studies, where it showed a very good safety profile. SAF has not been studied in de novo PD patients, and its potential positive effect on dyskinesia deserves further dedicated studies.

Physicochemical, pharmacokinetic and pharmacodynamic evaluations of novel ternary solid dispersion of rebamipide with poloxamer 407.

This study was conducted primarily to improve the solubility of rebamipide, a poorly water-soluble anti-ulcer drug, using novel ternary solid dispersion (SD) systems and secondly to evaluate the effect of solubility enhancement on its pharmacokinetic (PK) and pharmacodynamic (PD) profile. After dissolving the three components in aqueous medium, ternary SD containing the drug, sodium hydroxide (NaOH) and PVP-VA 64 was achieved by spray drying method, which was used as primary SD. Poloxamer 407, a surfactant polymer, was incorporated in this primary SD by four different methods: co-grinding, physical mixing, melting or spray drying. SD was then characterized by dissolution test, differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD) and Fourier transform infrared spectroscopy (FT-IR). The spray dried SD of poloxamer 407 together with primary SD displayed highest dissolution rate of the drug of about 70% after 2 h. DSC, PXRD and FT-IR characterized the amorphous state and molecular dispersion of the drug in the SD. PK and PD studies in Sprague-Dawley rats revealed that the bioavailability of the drug using optimal SD was about twofold higher than that of reference product, and the irritation area of stomach was significantly reduced in the ulcer-induced rat model using optimal SD as compared to the reference product.

QT effect of semagacestat at therapeutic and supratherapeutic doses.

OBJECTIVE: This thorough QT/ QT interval corrected for heart rate (QTc) study was designed to assess the potential of semagacestat, a functional gamma-secretase inhibitor, to delay cardiac repolarization. METHODS: In this Phase I, single-dose, randomized, 4-period crossover study, semagacestat was compared with placebo in 54 healthy male and female subjects between the ages of 19 and 63 years, inclusive. Each study period included single oral-dose administrations of semagacestat 140 mg, semagacestat 280 mg, moxifloxacin 400 mg, or placebo. Study subjects and the investigator were blinded to the identity of semagacestat and placebo; however, moxifloxacin was administered as open-label. Moxifloxacin was compared with placebo for assay sensitivity analysis. Pharmacokinetic parameters were also assessed. RESULTS: For each QTc, the upper bound of the 2-sided 90% confidence interval (CI) for the least squares mean difference between semagacestat (at both the 140- and 280-mg dose levels) and placebo was < 10 msec at all time points, and thus, within the limits set for clinical relevance in regulatory guidelines. CONCLUSIONS: The results of this study indicate that single doses of 140 and 280 mg semagacestat did not prolong QTc to a clinically significant degree.

Quantitative pharmacokinetic/pharmacodynamic analyses suggest that the 129/SVE mouse is a suitable preclinical pharmacology model for identifying small-molecule γ-secretase inhibitors.

Alzheimer's disease (AD) poses a serious public health threat to the United States. Disease-modifying drugs slowing AD progression are in urgent need, but they are still unavailable. According to the amyloid cascade hypothesis, inhibition of ß- or γ-secretase, key enzymes for the production of amyloid ß (Aß), may be viable mechanisms for the treatment of AD. For the discovery of γ-secretase inhibitors (GSIs), the APP-overexpressing Tg2576 mouse has been the preclinical model of choice, in part because of the ease of detection of Aß species in its brain, plasma, and cerebrospinal fluid (CSF). Some biological observations and practical considerations, however, argue against the use of the Tg2576 mouse. We reasoned that an animal model would be suitable for GSI discovery if the pharmacokinetic (PK)/pharmacodynamic (PD) relationship of a compound for Aß lowering in this model is predictive of that in human. In this study, we assessed whether the background 129/SVE strain is a suitable preclinical pharmacology model for identifying new GSIs by evaluating the translatability of the intrinsic PK/PD relationships for brain and CSF Aß across the Tg2576 and 129/SVE mouse and human. Using semimechanistically based PK/PD modeling, our analyses indicated that the intrinsic PK/PD relationship for brain Aßx-42 and CSF Aßx-40 in the 129/SVE mouse is indicative of that for human CSF Aß. This result, in conjunction with practical considerations, strongly suggests that the 129/SVE mouse is a suitable model for GSI discovery. Concurrently, the necessity and utilities of PK/PD modeling for rational interpretation of Aß data are established.

Preclinical pharmacokinetics and in vitro metabolism of brivanib (BMS-540215), a potent VEGFR2 inhibitor and its alanine ester prodrug brivanib alaninate.

PURPOSE: Brivanib alaninate is a prodrug of brivanib (BMS-540215), a potent oral VEGFR-2 inhibitor and is currently in development for the treatment of hepatocellular and colon carcinomas. In vitro and in vivo studies were conducted to characterize the preclinical pharmacokinetics and disposition of brivanib and brivanib alaninate, and antitumor efficacy in mice bearing human xenografts. METHODS: In vitro studies were conducted in liver and intestinal fractions, plasma and Caco-2 cells to assess the metabolic stability. Pharmacokinetics of brivanib were determined in preclinical species after administration of single intravenous or oral doses of both brivanib and brivanib alaninate. The antitumor efficacy was assessed at equimolar doses in nude mice bearing human tumor xenografts. Human efficacious dose was predicted based on projected human pharmacokinetic parameters and exposure at efficacious doses in the mouse efficacy models. RESULTS: In vitro and in vivo studies indicated that brivanib alaninate was efficiently converted to brivanib. Brivanib showed good brain penetration in rats consistent with its high intrinsic permeability and lack of active efflux in Caco-2 cells. The oral bioavailability of brivanib varied among species (22-88%) and showed dissolution rate-limited absorption even when combined with organic co-solvents. Administration of brivanib as brivanib alaninate allowed completely aqueous vehicles, and an improvement in the oral bioavailability (55-97%) was observed. The clearance of brivanib in humans is anticipated to be low to intermediate (hepatic extraction ratio < 0.7), while its volume of distribution is expected to be high. The minimum efficacious dose of brivanib alaninate was determined to be 60 mg/kg per day. CONCLUSIONS: Brivanib alaninate is rapidly and efficiently converted to the parent, brivanib, as demonstrated both in vitro and in vivo and offers an excellent mode to deliver brivanib orally.

Managlinat dialanetil, a fructose-1,6-bisphosphatase inhibitor for the treatment of type 2 diabetes.

Metabasis Therapeutics Inc (formerly Gensia Sicor Inc, which became SICOR Inc), in collaboration with Daiichi Sankyo Co Ltd (formerly Sankyo Co Ltd) is developing the fructose-1,6-bisphosphatase inhibitor managlinat dialanetil, a low-molecular weight purine nucleotide analog that inhibits gluconeogenesis, for the potential treatment of type 2 diabetes. Phase II clinical trials of the compound are underway in Europe and the US.

Interaction between murine spf-ash mutation and genetic background yields different metabolic phenotypes.

The spf-ash mutation in mice results in reduced hepatic and intestinal ornithine transcarbamylase. However, a reduction in enzyme activity only translates in reduced ureagenesis and hyperammonemia when an unbalanced nitrogen load is imposed. Six-week-old wild-type control and spf-ash mutant male mice from different genetic backgrounds (B6 and ICR) were infused intravenously with [(13)C(18)O]urea, l-[(15)N(2)]arginine, l-[5,5 D(2)]ornithine, l-[6-(13)C, 4,4,5,5, D(4)]citrulline, and l-[ring-D(5)]phenylalanine to investigate the interaction between genetic background and spf-ash mutation on ureagenesis, arginine metabolism, and nitric oxide production. ICR(spf-ash) mice maintained ureagenesis (5.5 +/- 0.3 mmol.kg(-1).h(-1)) and developed mild hyperammonemia (145 +/- 19 micromol/l) when an unbalanced nitrogen load was imposed; however, B6(spf-ash) mice became hyperammonemic (671 +/- 15 micromol/l) due to compromised ureagenesis (3.4 +/- 0.1 mmol.kg(-1).h(-1)). Ornithine supplementation restored ureagenesis and mitigated hyperammonemia. A reduction in citrulline entry rate was observed due to the mutation in both genetic backgrounds (wild-type: 128, spf-ash: 60; SE 4.0 micromol.kg(-1).h(-1)). Arginine entry rate was only reduced in B6(spf-ash) mice (B6(spf-ash): 332, ICR(spf-ash): 453; SE 20.6 micromol.kg(-1).h(-1)). Genetic background and mutation had an effect on nitric oxide production (B6: 3.4, B6(spf-ash): 2.8, ICR: 9.0, ICR(spf-ash): 4.6, SE 0.7 micromol.kg(-1).h(-1)). Protein breakdown was the main source of arginine during the postabsorptive state and was higher in ICR(spf-ash) than in B6(spf-ash) mice (phenylalanine entry rate 479 and 327, respectively; SE 18 micromol.kg(-1).h(-1)). Our results highlight the importance of the interaction between mutation and genetic background on ureagenesis, arginine metabolism, and nitric oxide production. These observations help explain the wide phenotypic variation of ornithine transcarbamylase deficiency in the human population.

The uptake of amino acids by the cyanobacterium Planktothrix rubescens is stimulated by light at low irradiances.

The rates of uptake of five amino acids--alanine, glutamate, glycine, leucine and serine--by axenic cultures of the cyanobacterium Planktothrix rubescens were measured over a range of irradiances using the (14)C-labelled amino acids at the nanomolar concentrations observed in Lake Zürich. The rates in the light exceeded the dark rates by as much as two- to ninefold. The light-affinity constants for stimulation were similar, indicating a similar process for each of the five amino acids. The E(k) (light saturation irradiance) for light stimulation was only 1 micromol m(-2) s(-1), less than the compensation point for photosynthesis and autotrophic growth, and much lower than the E(k) for either process. The E(k) for amino acid uptake was also less than the irradiance at which filaments obtain neutral buoyancy, which determines the depth at which they stratify and the irradiance they receive. This indicates that stimulation of amino acid uptake by light of low irradiances provides a mechanism for supplementing growth of filaments stratifying deep in the metalimnion, which, while able to grow at low irradiances, are often left with insufficient light to sustain them. Acetate uptake was also stimulated by light, but the kinetics differed.

Oral absorption and pharmacokinetics of rebamipide and rebamipide lysinate in rats.

Rebamipide is an anti-ulcer agent exhibiting a low aqueous solubility and a poor oral bioavailability. This study was conducted to examine if the rebamipide lysinate salt form would exhibit improved solubility profiles and higher oral bioavailability compared with rebamipide free acid. Both compounds showed pH-dependent solubility profiles, with the solubility of rebamipide lysinate dramatically improved at a median pH of 5.1 (17-fold increases) over free acid, but the improvement in the solubility was not as pronounced in artificial gastric and intestinal fluids (1.4- and 1.9-fold increases, respectively). The Cl, V(ss) and t1/2 in rats after i.v. injection of rebamipide (0.5 mg/kg) averaged 21.0 +/- 3.2 ml/min/kg, 0.3 +/- 0.0 L/kg, and 0.4 +/- 0.1 hr, respectively. No significant difference was observed in these parameters between rebamipide and rebamipide lysinate. Despite improved solubility profiles, the absolute oral bioavailability of rebamipide lysinate was not increased (5.1 vs. 4.8%) nor did AUC (407.8 vs. 383.6 ng x hr/ml) and C(max) (87.4 vs. 77.0 ng/ml) compared with rebamipide free acid. Rebamipide lysinate, however, showed a more rapid absorption, and initial serum drug concentrations were higher than those found for rebamipide free acid.

Development of suppository formulation safely improving rectal absorption of rebamipide, a poorly absorbable drug, by utilizing sodium laurate and taurine.

To develop the safe formulation that can safely improve bioavailability of poorly absorbable drugs and that is practically available, we prepared the suppositories of rebamipide, a poorly soluble and poorly absorbable antiulcer drug, by employing the combinatorial use of sodium laurate (C12), an absorption enhancer, with taurine (Tau) or L-glutamine (L-Gln), an adjuvant exerting the cytoprotective action. Although the dissolution of rebamipide from fatty base (FB) suppository prepared using Witepsol H-15 was very slow, it was remarkably improved by the addition of C12 and L-Gln or Tau into the suppository. On the other hand, the dissolution of rebamipide from water-soluble base (WB) suppository prepared using polyethylene glycol was very rapid and the addition of adjuvants did not influence its dissolution so much. Rectal absorption of rebamipide examined in rats was remarkably improved by FB suppository containing C12 or both C12 and Tau, while the enhancing effect of C12 was relatively small in the case of WB suppositories. Biochemical and histopathological studies have confirmed that FB suppository containing both C12 and Tau or L-Gln did not cause any serious local damage, while FB suppository containing C12 only caused the erosion and shrinkage for a lot of rectal epithelial cells. In conclusion, FB suppository employing the combinatorial use of C12 with Tau could be a promising formulation that is effective and safe enough for poorly absorbable drugs to be practically administered.

Metabolic response of muscle to alanine, glutamine, and valine supplementation during severe illness.

BACKGROUND: Alanine and glutamine are released from muscle in response to critical illness. Subsequent depletion of glutamine from muscle is proposed as a principal factor in the limitation of muscle protein synthesis in severely ill patients. The objective of this study was to assess the peripheral metabolic response to enteral supplementation of alanine, glutamine, and valine in critically ill patients. METHODS: Isotopic tracers of alanine, glutamine, and phenylalanine were given IV to 6 critically ill patients and 6 healthy volunteers. Blood sampling from the femoral artery and vein along with muscle biopsies provided assessment of leg (ie, muscle) kinetics. Measurements were obtained during enteral nutrition alone and then with combined alanine (11.25 g), glutamine (7.5 g) and valine (11.25 g) supplementation for 3 hours. RESULTS: Compared with healthy volunteers, critically ill patients had significantly reduced concentrations of alanine and glutamine in arterial plasma (p < .05), which increased significantly with amino acid supplementation. Muscle glutamine concentrations were significantly less in the patients and were not significantly affected by supplementation. Alanine and glutamine transport into and out of muscle and the rates of alanine and glutamine incorporation into and production from muscle were not affected by supplementation. Phenylalanine kinetics, as a marker of muscle protein metabolism, were not significantly altered by alanine, glutamine, and valine intake. CONCLUSIONS: These results demonstrate that alanine, glutamine, and valine administration fails to significantly affect muscle glutamine availability or muscle protein metabolism. These findings suggest that accelerated muscle catabolism in critically ill patients is not in response to any deficiency in alanine or glutamine availability.

Combinatorial use of sodium laurate with taurine or L-glutamine enhances colonic absorption of rebamipide, poorly absorbable antiulcer drug, without any serious histopathological mucosal damages.

We previously reported that the combinatorial use of sodium laurate (C12) with several amino acids such as taurine (Tau) and L-glutamine (L-Gln) enhanced the colonic absorption of phenol red with attenuating the local toxicity caused by C12. However, even these amino acids could not protect epithelial cells from being damaged if the mucosal damage got worse to the coagulation necrosis by an excessive dose of C12. Comparing C12 with sodium caprate (C10), used in drug products marketed, 100 micromol C10 was needed to exert the similar absorption-enhancement of rebamipide, a poorly absorbable antiulcer drug, to that by 10 micromol C12, and 100 micromol C10 was obviously more toxic to the mucosa than 10 micromol C12. The combinatorial use of C12 with Tau or L-Gln enhanced the colonic absorption of rebamipide four to nine times larger in AUC than the control. Histopathologic studies clearly showed that Tau and L-Gln exerted the cytoprotective action on epithelial cells suffering from slight damages such as shrinkage and exfoliation, more articulately at 6 h than at 1.5 h after dosing. In conclusion, the combinatorial use of C12 with Tau or L-Gln could lead to a novel formulation improving the bioavailability of poorly absorbable drugs without any serious local damages.

The simultaneous biosynthesis and uptake of amino acids by Lactococcus lactis studied by (13)C-labeling experiments.

Uniformly (13)C labeled glucose was fed to a lactic acid bacterium growing on a defined medium supplemented with all proteinogenic amino acids except glutamate. Aspartate stemming from the protein pool and from the extracellular medium was enriched with (13)C disclosing a substantial de novo biosynthesis of this amino acid simultaneous to its uptake from the growth medium and a rapid exchange flux of aspartate over the cellular membrane. Phenylalanine, alanine, and threonine were also synthesized de novo in spite of their presence in the growth medium.

NMR spectroscopic study on the metabolic fate of [3-(13)C]alanine in astrocytes, neurons, and cocultures: implications for glia-neuron interactions in neurotransmitter metabolism.

Nuclear magnetic resonance (NMR) spectroscopy and biochemical assays were used to study the fate of [3-(13)C]alanine in astrocytes, neurons, and cocultures. (1)H- and (13)C-NMR analysis of the media demonstrated a high and comparable uptake of [3-(13)C]alanine by the cells. Thereafter, alanine is transaminated predominantly to [3-(13)C]pyruvate, from which the (13)C-label undergoes different metabolic pathways in astrocytes and neurons: Lactate is almost exclusively synthesized in astrocytes, while in neurons and cocultures labeled neurotransmitter amino acids are formed, i.e., glutamate and gamma-aminobutyric acid (GABA). A considerable contribution of the anaplerotic pathway is observed in cocultures, as concluded from the ratio (C-2-C-3)/C-4 of labeled glutamine. Analysis of the multiplet pattern of glutamate isotopomers indicates carbon scrambling through the TCA cycle and the use of alanine also as energy substrate in neurons. In cocultures, astrocyte-deduced lactate and unlabeled exogenous carbon substrates contribute to glutamate synthesis and dilute the [2-(13)C]acetyl-CoA pool by 30%. The coupling of neuronal activity with shuttling of tricarboxylic acid (TCA) cycle-derived metabolites between astrocytes and neurons is concluded from the use of [4-(13)C]-monolabeled glutamate leaving the first TCA cycle turn already for glutamine and GABA synthesis, as well as from the labeling pattern of extracellular glutamine. Further evidence of a metabolic interaction between astrocytes and neurons is obtained, as alanine serves as a carbon and nitrogen carrier through the synthesis and regulated release of lactate from astrocytes for use by neurons. Complementary to the glutamine-glutamate cycle in the brain, a lactate-alanine shuttle between astrocytes and neurons would account for the nitrogen exchange of the glutamatergic neurotransmitter cycle in mammalian brain.