2 results
2235 15N-Leucine transport across the blood brain barrier is significantly impaired in the glutamine synthetase-inhibited brain
- Shaun E. Gruenbaum, Roni Dhaher, Kevin Behar, Hitten Zaveri, Mark Erfe, Tore Eid
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- Journal:
- Journal of Clinical and Translational Science / Volume 2 / Issue S1 / June 2018
- Published online by Cambridge University Press:
- 21 November 2018, p. 1
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OBJECTIVES/SPECIFIC AIMS: Astroglial glutamine synthetase (GS), which metabolizes glutamate and ammonia to glutamine, is critical for the detoxification of brain ammonia, clearance of synaptic glutamate, and production of brain glutamine. Perturbations in the expression and activity of GS are thought to play a causative role in the pathogenesis of several conditions of abnormal neurotransmission. Although the long-term consequences of GS inhibition on amino acid homeostasis in the brain are unknown, it is thought that amino acid influx in the brain is tightly coupled with glutamine efflux via the L-type amino acid transporter. Both glutamine and leucine serve many critical functions in the brain including protein synthesis, gene expression, insulin regulation, and immune signaling. The objective of this study was to determine the effects of chronic GS inhibition with methionine sulfoximine (MSO) on glutamine and leucine homeostasis in the brain. METHODS/STUDY POPULATION: In total, 12 rats were surgically implanted with microdialysis guide cannulas in the bilateral dentate gyrus. Rats were randomly divided for surgical implantation of either a MSO (n=6) or phosphate buffer saline (PBS; n=6) pump in the right dentate gyrus. After 7 days, bilateral microdialysis probes were placed under brief isoflurane anesthesia, and microdialysis flow was established by infusing 0.5 µL/min of artificial extracellular fluid. Dialysate samples were collected every 30 minutes for the duration of the experiment. A 113 mM 15N-Leucine (3.6 mL/h) and 2 M 2–13C-sodium acetate (0.0633 μL/g/min for t=0–5 min, 0.0316 μL/g/min for t=5–10 min, and 0.0253 μL/g/min for t>10 min) solution was infused intravenously for 300 minutes. The EZ:Faast Free Amino Acid analysis kit and ultra-performance liquid chromatography/tandem mass spectrometry was used for quantification of amino acids in the dialysate fluid. RESULTS/ANTICIPATED RESULTS: At baseline (t=0 h), the concentrations of glutamine were significantly lower in MSO-treated rats (p<0.001) in the ipsilateral (GS-inhibited) hippocampus. There were no differences in glutamine concentrations between MSO and PBS-treated rats in the contralateral hippocampus. In PBS-treated rats, there was a significant increase in 15N-leucine between t=0 hour and t=5 hour in the contralateral (p<0.05) and ipsilateral (p<0.05) hippocampus. In MSO-treated rats, there was a significant increase in 15N-leucine between t=0 and t=5 hours in the contralateral (p<0.05) hippocampus, but not in the ipsilateral hippocampus (p=ns.). DISCUSSION/SIGNIFICANCE OF IMPACT: This study demonstrated for the first time that basal glutamine concentrations are low in areas of the brain where GS is acutely inhibited, and that leucine uptake in these brain areas are markedly decreased. Perturbations in glutamine and leucine homeostasis have been implicated in several disease processes including diabetes, obesity, liver disease, immune system dysfunction, epilepsy, and cancer, and the glutamine-dependent leucine influx in the brain may be a novel and important therapeutic target to treat these conditions.
2092: Chronic branched-chain amino acid ingestion aggravates hilar neuron loss in a rodent model of temporal lobe epilepsy
- Shaun Evan Gruenbaum, Roni Dhaher, Amedeo Rapuano, Tore Eid
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- Journal:
- Journal of Clinical and Translational Science / Volume 1 / Issue S1 / September 2017
- Published online by Cambridge University Press:
- 10 May 2018, p. 56
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OBJECTIVES/SPECIFIC AIMS: We previously developed a translationally relevant model of temporal lobe epilepsy (TLE) in which glutamine synthetase is irreversibility inhibited by methionine sulfoximine (MSO), resulting in spontaneous seizures and dentate hilar neuron loss. The objective of this study was to determine the effects of chronic BCAA ingestion on neuronal viability in the dentate hilus in the MSO model of TLE. METHODS/STUDY POPULATION: Sixteen rats were randomly divided into 2 groups: 8 rats drank a 4% aqueous solution of all 3 BCAAs (BCAA group) ad libitum for 31 days, and the other 8 rats drank regular water (control group) for the same period. After 10 days of drinking, a microinfusion cannula (Alzet osmotic pump, model 2004) was surgically implanted in the right dentate gyrus to continuously infuse MSO at a rate of 0.625 g/hour for 28 days. After 31 days of drinking, rats were perfused transcardially with 0.9% NaCl followed by 4% paraformaldehyde in phosphate buffer. The brains were removed and fixed, sectioned on a Vibratome at 50-μm thickness, and were mounted on a gelatin-coated slides and stained with NeuN. Neuron counts in the hilar region were performed ipsilateral and contralateral to the infusion site using a stereological technique. RESULTS/ANTICIPATED RESULTS: Rats in the BCAA group had 37% fewer neurons in the ipsilateral dentate hilus than the control group (5.8×10−4±6.8×10−5 vs. 8.9×10−4±5.6×10−5 cells, respectively, p<0.01). Similarly, rats in the BCAA group had 39% fewer neurons in the contralateral dentate hilus than the control group (5.0×10−4±5.8×10−5 vs. 7.0×10−4±3.4×10−5 cells, respectively, p=0.01). DISCUSSION/SIGNIFICANCE OF IMPACT: This study demonstrates that chronic ingestion of BCAAs aggravates hilar neuronal loss in a translationally relevant rodent model of MTLE. This study gives important insight into how BCAAs may affect neuronal viability. Although the role of BCAAs on seizure activity is poorly understood, these results suggest that BCAAs may play an important role in neurochemical modulation and neurotoxicity.