Large Neutral Amino Acid Transporter Research Articles

Possibility of Amino Acid Treatment to Prevent the Psychiatric Disorders via Modulation of the Production of Tryptophan Metabolite Kynurenic Acid.

Kynurenic acid, a metabolite of the kynurenine pathway of tryptophan catabolism, acts as an antagonist for both the α7 nicotinic acetylcholine receptor and glycine coagonist sites of the N-methyl-d-aspartic acid receptor at endogenous brain concentrations. Elevation of brain kynurenic acid levels reduces the release of neurotransmitters such as dopamine and glutamate, and kynurenic acid is considered to be involved in psychiatric disorders such as schizophrenia and depression. Thus, the control of kynurenine pathway, especially kynurenic acid production, in the brain is an important target for the improvement of brain function or the effective treatment of brain disorders. Astrocytes uptake kynurenine, the immediate precursor of kynurenic acid, via large neutral amino acid transporters, and metabolize kynurenine to kynurenic acid by kynurenine aminotransferases. The former transport both branched-chain and aromatic amino acids, and the latter have substrate specificity for amino acids and their metabolites. Recent studies have suggested the possibility that amino acids may suppress kynurenic acid production via the blockade of kynurenine transport or via kynurenic acid synthesis reactions. This approach may be useful in the treatment and prevention of neurological and psychiatric diseases associated with elevated kynurenic acid levels.

Adaptive responses to maternal nutrient restriction alter placental transport in ewes

IntroductionMaternal nutrient partitioning, uteroplacental blood flow, transporter activity, and fetoplacental metabolism mediate nutrient delivery to the fetus. Inadequate availability or delivery of nutrients results in intrauterine growth restriction (IUGR), a leading cause of neonatal morbidity and mortality. Maternal nutrient restriction can result in IUGR, but only in an unforeseeable subset of individuals. MethodsTo elucidate potential mechanisms regulating fetal nutrient availability, singleton sheep pregnancies were generated by embryo transfer. Pregnant ewes received either a 50% NRC (NR; n = 24) or 100% NRC (n = 7) diet from gestational Day 35 until necropsy on Day 125. Maternal weight did not correlate with fetal weight; therefore, the six heaviest (NR Non-IUGR) and five lightest (NR IUGR) fetuses from nutrient-restricted ewes, and seven 100% NRC fetuses, were compared to investigate differences in nutrient availability. ResultsInsulin, multiple amino acids, and their metabolites, were reduced in fetal circulation of NR IUGR compared to NR Non-IUGR and 100% NRC pregnancies. In contrast, glucose in fetal fluids was not different between groups. There was a nearly two-fold reduction in placentome volume and fetal/maternal interface length in NR IUGR compared to NR Non-IUGR and 100% NRC pregnancies. Changes in amino acid concentrations were associated with altered expression of cationic (SLC7A2, SLC7A6, and SLC7A7) and large neutral (SLC38A2) amino acid transporters in placentomes. DiscussionResults establish a novel approach to study placental adaptation to maternal undernutrition in sheep and support the hypothesis that amino acids and polyamines are critical mediators of placental and fetal growth in sheep.

Targeted tumour theranostics in mice via carbon quantum dots structurally mimicking large amino acids.

Strategies for selectively imaging and delivering drugs to tumours typically leverage differentially upregulated surface molecules on cancer cells. Here, we show that intravenously injected carbon quantum dots, functionalized with multiple paired α-carboxyl and amino groups that bind to the large neutral amino acid transporter 1 (which is expressed in most tumours), selectively accumulate in human tumour xenografts in mice and in an orthotopic mouse model of human glioma. The functionalized quantum dots, which structurally mimic large amino acids and can be loaded with aromatic drugs through π–π stacking interactions, enabled—in the absence of detectable toxicity—near-infrared fluorescence and photoacoustic imaging of the tumours and a reduction in tumour burden after the targeted delivery of chemotherapeutics to the tumours. The versatility of functionalization and high tumour selectivity of the quantum dots make them broadly suitable for tumour-specific imaging and drug delivery.

Endothelial-derived plasma exosome proteins in Alzheimer's disease angiopathy.

Small cerebral vascular disease (SCeVD) demonstrated by white matter hyperintensity (WMH) on MRI contributes to the development of dementia in Alzheimer's disease (AD), but it has not been possible to correlate onset, severity, or protein components of SCeVD with characteristics of WMH in living patients. Plasma endothelial-derived exosomes (EDEs) were enriched by two-step immunoabsorption from four groups of participants with no clinical evidence of cerebrovascular disease: cognitively normal (CN) without WMH (CN without SCeVD, n=20), CN with SCeVD (n=22), preclinical AD (pAD)+mild cognitive impairment (MCI) without SCeVD (pAD/MCI without SCeVD, n=22), and pAD/MCI with SCeVD (n=16) for ELISA quantification of cargo proteins. Exosome marker CD81-normalized EDE levels of the cerebrovascular-selective biomarkers large neutral amino acid transporter 1 (LAT-1), glucose transporter type 1 (Glut-1), and permeability-glycoprotein (p-GP, ABCB1) were similarly significantly higher in the CN with SCeVD and pAD/MCI with SCeVD groups than their corresponding control groups without SCeVD. CD81-normalized EDE levels of Aβ40 and Aβ42 were significantly higher in the pAD/MCI with SCeVD group but not in the CN with SCeVD group relative to controls without SCeVD. Levels of normal cellular prion protein (PrPc), a receptor for amyloid peptides, and phospho-181T-tau were higher in both CN and pAD/MCI with SCeVD groups than in the corresponding controls. High EDE levels of Aβ40, Aβ42, and phospho-181T-tau in patients with WMH suggesting SCeVD appear at the pre-clinical or MCI stage of AD and therapeutic lowering of neurotoxic peptide levels may delayprogression of ADangiopathy.

Improvement, cloning, and expression of an in silico designed protein enriched with large neutral amino acids in Pichia pastoris for possible application in phenylketonuria.

Phenylketonuria (PKU) is an inborn disease caused by defective phenylalanine hydroxylase, which consequently results in the accumulation of phenylalanine in the brain leading to further complications. One of the promising approaches in dietary treatment is the supplementation of large neutral amino acid (LNAA). The LNAA compete with phenylalanine for the common L-type LNAA transporter across the blood-brain barrier, and decrease phenylalanine levels in the brain. In this study, the earlier LNAA-enriched protein model was improved (Protein Model-66) and validated in silico. The reverse translated and codon-optimized synthetic LNAA66 gene was cloned into pPICZαC and expressed in Pichia pastoris. The expressed protein was purified by His Select affinity chromatography. SDS-PAGE and Western blotting analysis showed a band at an expected molecular weight of 12kDa, confirming the expression of the modeled protein. To our knowledge, this is the first report showing the cloning and expression of an in silico designed LNAA-enriched protein. PRACTICAL APPLICATIONS: One of the promising dietary treatment of phenylketonuria (PKU) is the supplementation of large neutral amino acid (LNAA), wherein high levels of LNAA compete with phenylalanine for the same L-type LNAA transporter, and consequently decrease phenylalanine accumulation in the brain, thereby decreasing neurological complications. For the first time, here, we are showing that an in silico designed and validated Protein Model-66, rich in LNAA, can be successfully cloned and expressed in Pichia pastoris. The complete biochemical and structural characterization of this protein will give a clear insight into its potential application for PKU treatment. The protein can be potentially used as a supplement to treat PKU to those who are non-adherent to the restricted, non-palatable, and expensive diet. Furthermore, this novel and effective strategy of in silico designing, cloning and expression can be exploited to develop proteins for various applications of industrial, food, medical, and academic relevance.

Extraprostatic Uptake of 18F-Fluciclovine: Differentiation of Nonprostatic Neoplasms From Metastatic Prostate Cancer.

OBJECTIVE. Fluciclovine is a synthetic radiolabeled amino acid analog used for imaging of biochemical recurrent prostate cancer. Uptake of fluciclovine is mediated by several amino acid transporters, including alanine-serine-cysteine transporter 2 and large neutral amino acid transporters, which are known to be overexpressed in other malignancies. CONCLUSION. Knowledge of the common patterns of prostate cancer recurrence, in addition to what other neoplasms can show uptake, is critical for accurate study interpretation.

Drug Delivery Strategies to Overcome the Blood-Brain Barrier (BBB).

The brain capillary endothelium serves both as an exchange site for gases and solutes between blood and brain and as a protective fence against neurotoxic compounds from the blood. While this "blood-brain barrier" (BBB) function protects the fragile environment in the brain, it also poses a tremendous challenge for the delivery of drug compounds to the brain parenchyma. Paracellular brain uptake of drug compounds is limited by the physical tightness of the endothelium, which is tightly sealed with junction complexes. Transcellular uptake of lipophilic drug compounds is limited by the activity of active efflux pumps in the luminal membrane. As a result, the majority of registered CNS drug compounds are small lipophilic compounds which are not efflux transporter substrates. Small molecule CNS drug development therefore focuses on identifying compounds with CNS target affinity and modifies these in order to optimize lipophilicity and decrease efflux pump interactions. Since efflux pump activity is limiting drug uptake, it has been investigated whether coadministration of drug compounds with efflux pump inhibitors could increase drug uptake. While the concept works to some extent, a lot of challenges have been encountered in terms of obtaining efficient inhibition while avoiding adverse effects.Some CNS drug compounds enter the brain via nutrient transport proteins, an example is the levodopa, a prodrug of Dopamine, which crosses the BBB via the large neutral amino acid transporter LAT1. While carrier-mediated transport of drug compounds may seem attractive, the development of drugs targeting transporters is very challenging, since the compounds should have a good fit to the binding site, while still maintaining their CNS target affinity.Receptor-mediated transport of drug compounds, especially biotherapeutics, conjugated to a receptor-binding ligand has shown some promise, although the amounts transported are rather low. This also holds true for drug-conjugation to cell-penetrating peptides. Due to the low uptake of biotherapeutics, barrier-breaching approaches such as mannitol injections and focused ultrasound have been employed with some success to patient groups with no other treatment options.

The Role of Large Neutral Amino Acid Transporter (LAT1) in Cancer.

The solute carrier family 7 (SLC7) can be categorically divided into two subfamilies, the L-type amino acid transporters (LATs) including SLC7A5-13, and SLC7A15, and the cationic amino acid transporters (CATs) including SLC7A1-4 and SLC7A14. Members of the CAT family transport predominantly cationic amino acids by facilitating diffusion with intracellular substrates. LAT1 (also known as SLC7A5), is defined as a heteromeric amino acid transporter (HAT) interacting with the glycoprotein CD98 (SLC3A2) through a conserved disulfide to uptake not only large neutral amino acids, but also several pharmaceutical drugs to cells. In this review, we provide an overview of the interaction of the structure-function of LAT1 and its essential role in cancer, specifically, its role at the blood-brain barrier (BBB) to facilitate the transport of thyroid hormones, pharmaceuticals (e.g., I-DOPA, gabapentin), and metabolites into the brain. LAT1 expression increases as cancers progress, leading to higher expression levels in highgrade tumors and metastases. In addition, LAT1 plays a crucial role in cancer-associated reprogrammed metabolic networks by supplying tumor cells with essential amino acids. The increasing understanding of the role of LAT1 in cancer has led to an increase in interest surrounding its potential as a drug target for cancer treatment.

Characterization of the expression of LAT1 as a prognostic indicator and a therapeutic target in renal cell carcinoma

Large neutral amino acid transporter 1 (LAT1, SLC7A5) is abundantly expressed in various types of cancer, and it has been thought to assist cancer progression through its activity for uptake of neutral amino acids. However, the roles of LAT1 in renal cell carcinoma (RCC) prognosis and treatment remain uncharacterized. Therefore, we first retrospectively examined the LAT1 expression profile and its associations with clinical factors in RCC tissues (n = 92). The results of immunohistochemistry showed that most of the tissues examined (92%) had cancer-associated LAT1 expression. Furthermore, the overall survival (OS) and progression-free survival (PFS) were shorter in patients with high LAT1 expression levels than in those with low LAT1 expression levels (P = 0.018 and 0.014, respectively), and these associations were further strengthened by the results of univariate and multivariate analyses. Next, we tested the effects of JPH203, which is a selective LAT1 inhibitor, on RCC-derived Caki-1 and ACHN cells. It was found that JPH203 inhibited the growth of these cell types in a dose-dependent manner. Moreover, JPH203 clearly suppressed their migration and invasion activities. Thus, our results show that LAT1 has a great potential to become not only a prognosis biomarker but also a therapeutic target in RCC clinical settings.

Bioengineered miR-328-3p modulates GLUT1-mediated glucose uptake and metabolism to exert synergistic antiproliferative effects with chemotherapeutics

MicroRNAs (miRNAs or miRs) are small noncoding RNAs derived from genome to control target gene expression. Recently we have developed a novel platform permitting high-yield production of bioengineered miRNA agents (BERA). This study is to produce and utilize novel fully-humanized BERA/miR-328-3p molecule (hBERA/miR-328) to delineate the role of miR-328-3p in controlling nutrient uptake essential for cell metabolism. We first demonstrated successful high-level expression of hBERA/miR-328 in bacteria and purification to high degree of homogeneity (>98%). Biologic miR-328-3p prodrug was selectively processed to miR-328-3p to suppress the growth of highly-proliferative human osteosarcoma (OS) cells. Besides glucose transporter protein type 1, gene symbol solute carrier family 2 member 1 (GLUT1/SLC2A1), we identified and verified large neutral amino acid transporter 1, gene symbol solute carrier family 7 member 5 (LAT1/SLC7A5) as a direct target for miR-328-3p. While reduction of LAT1 protein levels by miR-328-3p did not alter homeostasis of amino acids within OS cells, suppression of GLUT1 led to a significantly lower glucose uptake and decline in intracellular levels of glucose and glycolytic metabolite lactate. Moreover, combination treatment with hBERA/miR-328 and cisplatin or doxorubicin exerted a strong synergism in the inhibition of OS cell proliferation. These findings support the utility of novel bioengineered RNA molecules and establish an important role of miR-328-3p in the control of nutrient transport and homeostasis behind cancer metabolism.

Transcerebral exchange kinetics of large neutral amino acids during acute inspiratory hypoxia in humans

Hypoxaemia is present in many critically ill patients, and may contribute to encephalopathy. Changes in the passage of large neutral amino acids (LNAAs) across the blood-brain barrier (BBB) with an increased cerebral influx of aromatic amino acids into the brain may concurrently be present and also contribute to encephalopathy, but it has not been established whether hypoxaemia per se may trigger such changes. We measured cerebral blood flow (CBF) in 11 healthy men using the Kety-Schmidt technique and obtained paired arterial and jugular-venous blood samples for the determination of LNAAs by high performance liquid chromatography at baseline and after 9 hours of poikilocapnic normobaric hypoxia (12% O2). Transcerebral net exchange was determined by the Fick principle, and transport of LNAAs across the BBB was determined mathematically. Hypoxia increased both the systemic and corresponding cerebral delivery of the aromatic amino acid phenylalanine, and the branched-chain amino acids leucine and isoleucine. Despite this, the transcerebral net exchange values and mathematically derived brain extracellular concentrations for all LNAAs were unaffected. In conclusion, the observed changes in circulating LNAAs triggered by hypoxaemia do not affect the transcerebral exchange kinetics of LNAAs to such an extent that their brain extracellular concentrations are affected.

Delayed access to feed alters expression of genes associated with carbohydrate and amino acid utilization in newly hatched broiler chicks.

Newly hatched chicks must transition from lipid-rich yolk to carbohydrate-rich feed as their primary nutrient source, and posthatch delays in access to feed can have long-term negative consequences on growth and metabolism. In this study, impacts of delayed access to feed at hatch on expression of genes related to nutrient uptake and utilization in two metabolically important tissues, liver and muscle, were determined in broiler (meat-type) chickens. Hatched chicks were given access to feed within 3 h (fed) or delayed access to feed for 48 h (delayed fed), and liver and breast muscle were collected from males at hatch and 4 h, 1 day, 2 days, 4 days, and 8 days posthatch for analysis of gene expression. Differential expression of carbohydrate response element-binding protein and peroxisome proliferator-activated receptor-γ in muscle and liver was observed, with results indicating a transitional delay from lipid to carbohydrate metabolism when hatched chicks were not given immediate access to feed. Extended upregulation of insulin receptor mRNA was observed in both tissues in delayed fed birds, suggesting increased sensitivity to circulating levels of the hormone. Developmental delays in expression patterns of cationic amino acid transporters 1 and 2 in both tissues and large neutral amino acid transporter 1 in muscle were also apparent when immediate feed access was prevented. These data suggest that delayed transition to carbohydrate use and altered nutrient transport and utilization within liver and breast muscle are key factors negatively affecting growth and metabolism following delayed feed access in broiler chickens.

Age- and disease-specific changes of the kynurenine pathway in Parkinson's and Alzheimer's disease.

The kynurenine (Kyn) pathway, which regulates neuroinflammation and N‐methyl‐d‐aspartate receptor activation, is implicated in Parkinson’s disease (PD) and Alzheimer’s disease (AD). Age‐related changes in Kyn metabolism and altered cerebral Kyn uptake along large neutral amino acid transporters, could contribute to these diseases. To gain further insight into the role and prognostic potential of the Kyn pathway in PD and AD, we investigated systemic and cerebral Kyn metabolite production and estimations of their transporter‐mediated uptake in the brain. Kyn metabolites and large neutral amino acids were retrospectively measured in serum and cerebrospinal fluid (CSF) of clinically well‐characterized PD patients (n = 33), AD patients (n = 33), and age‐matched controls (n = 39) using solid‐phase extraction‐liquid chromatographic‐tandem mass spectrometry. Aging was disease independently associated with increased Kyn, kynurenic acid and quinolinic acid in serum and CSF. Concentrations of kynurenic acid were reduced in CSF of PD and AD patients (p = 0.001; p = 0.002) but estimations of Kyn brain uptake did not differ between diseased and controls. Furthermore, serum Kyn and quinolinic acid levels strongly correlated with their respective content in CSF and Kyn in serum negatively correlated with AD disease severity (p = 0.002). Kyn metabolites accumulated with aging in serum and CSF similarly in PD patients, AD patients, and control subjects. In contrast, kynurenic acid was strongly reduced in CSF of PD and AD patients. Differential transporter‐mediated Kyn uptake is unlikely to majorly contribute to these cerebral Kyn pathway disturbances. We hypothesize that the combination of age‐ and disease‐specific changes in cerebral Kyn pathway activity could contribute to reduced neurogenesis and increased excitotoxicity in neurodegenerative disease.

The IDO Metabolic Trap Hypothesis for the Etiology of ME/CFS.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating noncommunicable disease brandishing an enormous worldwide disease burden with some evidence of inherited genetic risk. Absence of measurable changes in patients’ standard blood work has necessitated ad hoc symptom-driven therapies and a dearth of mechanistic hypotheses regarding its etiology and possible cure. A new hypothesis, the indolamine-2,3-dioxygenase (IDO) metabolic trap, was developed and formulated as a mathematical model. The historical occurrence of ME/CFS outbreaks is a singular feature of the disease and implies that any predisposing genetic mutation must be common. A database search for common damaging mutations in human enzymes produces 208 hits, including IDO2 with four such mutations. Non-functional IDO2, combined with well-established substrate inhibition of IDO1 and kinetic asymmetry of the large neutral amino acid transporter, LAT1, yielded a mathematical model of tryptophan metabolism that displays both physiological and pathological steady-states. Escape from the pathological one requires an exogenous perturbation. This model also identifies a critical point in cytosolic tryptophan abundance beyond which descent into the pathological steady-state is inevitable. If, however, means can be discovered to return cytosolic tryptophan below the critical point, return to the normal physiological steady-state is assured. Testing this hypothesis for any cell type requires only labelled tryptophan, a means to measure cytosolic tryptophan and kynurenine, and the standard tools of tracer kinetics.

Development of LAT‐1 inhibitors for the treatment of pancreatic cancers

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer and represents one of the world's most deadly cancers with a five year survival of only 8%. As such there is a desperate need to develop effective new medicines for pancreatic cancers. The large neutral aminoacid transporter 1 (LAT‐1) is highly expressed in PDAC tumors and provides a conduit for these tumors to acquire essential amino acids like leucine and methionine. Beyond their incorporation into proteins, leucine is an important regulator of mTOR signaling and methionine is essential for one carbon metabolism and polyamine biosynthesis. Targeting LAT‐1 inhibits the uptake of these essential building blocks and inhibits cell growth.There has been significant interest in developing LAT‐1 inhibitors for oncology applications and virtually all of the known LAT‐1 inhibitors are predicated upon a phenylalanine scaffold. Here, we showed that piperazine‐based materials effectively inhibited leucine and methionine import into L3.6pl human pancreatic cancer cells. We also demonstrated that our lead LAT‐1 inhibitor resulted in a dose‐dependent decrease in intracellular methionine, leucine, and depleted polyamine pools in L3.6pl cells in vitro. Moreover, these treated cells were not rescued by exogenous polyamines indicating that polyamine import could not revive the treated cells. Our different molecular design provides new opportunities for enhanced targeting and both the synthesis and bioevaluation of these new LAT‐1 inhibitor designs will be discussed.Support or Funding InformationUCF College of Medicine (#25014202)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Discovery of Potent Inhibitors for the Large Neutral Amino Acid Transporter 1 (LAT1) by Structure-Based Methods

The large neutral amino acid transporter 1 (LAT1) is a promising anticancer target that is required for the cellular uptake of essential amino acids that serve as building blocks for cancer growth and proliferation. Here, we report a structure-based approach to identify chemically diverse and potent inhibitors of LAT1. First, a homology model of LAT1 that is based on the atomic structures of the prokaryotic homologs was constructed. Molecular docking of nitrogen mustards (NMs) with a wide range of affinity allowed for deriving a common binding mode that could explain the structure−activity relationship pattern in NMs. Subsequently, validated binding hypotheses were subjected to molecular dynamics simulation, which allowed for extracting a set of dynamic pharmacophores. Finally, a library of ~1.1 million molecules was virtually screened against these pharmacophores, followed by docking. Biological testing of the 30 top-ranked hits revealed 13 actives, with the best compound showing an IC50 value in the sub-μM range.

L-DOPA functionalized, multi-branched gold nanoparticles as brain-targeted nano-vehicles

The blood–brain barrier (BBB) is a protective endothelial barrier lining the brain microvasculature which prevents brain delivery of therapies against brain diseases. Hence, there is an urgent need to develop vehicles which efficiently penetrate the BBB to deliver therapies into the brain. The drug L-DOPA efficiently and specifically crosses the BBB via the large neutral amino acid transporter (LAT)-1 protein to enter the brain. Thus, we synthesized L-DOPA-functionalized multi-branched nanoflower-like gold nanoparticles (L-DOPA-AuNFs) using a seed-mediated method involving catechols as a direct reducing-cum-capping agent, and examined their ability to cross the BBB to act as brain-penetrating nanovehicles. We show that L-DOPA-AuNFs efficiently penetrate the BBB compared to similarly sized and shaped AuNFs functionalized with a non-targeting ligand. Furthermore, we show that L-DOPA-AuNFs are efficiently internalized by brain macrophages without inducing inflammation. These results demonstrate the application of L-DOPA-AuNFs as a non-inflammatory BBB-penetrating nanovehicle to efficiently deliver therapies into the brain.

Dysregulated Handling of Dietary Protein and Muscle Protein Synthesis After Mixed-Meal Ingestion in Maintenance Hemodialysis Patients

IntroductionSkeletal muscle loss is common in patients with renal failure who receive maintenance hemodialysis (MHD) therapy. Regular ingestion of protein-rich meals are recommended to help offset muscle protein loss in MHD patients, but little is known about the anabolic potential of this strategy.MethodsEight MHD patients (age: 56 ± 5 years; body mass index [BMI]: 32 ± 2 kg/m2) and 8 nonuremic control subjects (age: 50 ± 2 years: BMI: 31 ± 1 kg/m2) received primed continuous L-[ring-2H5]phenylalanine and L-[1-13C]leucine infusions with blood and muscle biopsy sampling on a nondialysis day. Participants consumed a mixed meal (546 kcal; 20-g protein, 59-g carbohydrates, 26-g fat) with protein provided as L-[5,5,5-2H3]leucine-labeled eggs.ResultsCirculating dietary amino acid availability was reduced in MHD patients (41 ± 5%) versus control subjects (61 ± 4%; P = 0.03). Basal muscle caspase-3 protein content was elevated (P = 0.03) and large neutral amino acid transporter 1 (LAT1) protein content was reduced (P = 0.02) in MHD patients versus control subjects. Basal muscle protein synthesis (MPS) was ∼2-fold higher in MHD patients (0.030 ± 0.005%/h) versus control subjects (0.014 ± 0.003%/h) (P = 0.01). Meal ingestion failed to increase MPS in MHD patients (absolute change from basal: 0.0003 ± 0.007%/h), but stimulated MPS in control subjects (0.009 ± 0.002%/h; P = 0.004).ConclusionsMHD patients demonstrated muscle anabolic resistance to meal ingestion. This blunted postprandial MPS response in MHD patients might be related to high basal MPS, which results in a stimulatory ceiling effect and/or reduced plasma dietary amino acid availability after mixed-meal ingestion.

LAT1-like transporters regulate dopaminergic transmission and sleep in Drosophila.

Amino acid transporters are involved in functions reportedly linked to the sleep/wake cycle: neurotransmitter synthesis and recycling, the regulation of synaptic strength, protein synthesis, and energy metabolism. In addition, the existence of bidirectional relationships among extracellular content, transport systems, and sleep/wake states is receiving emerging support. Nevertheless, the connection between amino acid transport and sleep/wake regulation remains elusive. To address this question, we used Drosophila melanogaster and investigated the role of LAT1 (large neutral amino acid transporter 1) transporters. We show that the two Drosophila LAT1-like transporters: Juvenile hormone Inducible-21 and minidiscs (Mnd) are required in dopaminergic neurons for sleep/wake regulation. Down-regulating either gene in dopaminergic neurons resulted in higher daily sleep and longer sleep bout duration during the night, suggesting a defect in dopaminergic transmission. Since LAT1 transporters can mediate in mammals the uptake of L-DOPA, a precursor of dopamine, we assessed amino acid transport efficiency by L-DOPA feeding. We find that downregulation of JhI-21, but not Mnd, reduced the sensitivity to L-DOPA as measured by sleep loss. JhI-21 downregulation also attenuated the sleep loss induced by continuous activation of dopaminergic neurons. Since LAT1 transporters are known to regulate target of rapamycin (TOR) signaling, we investigated the role of this amino acid sensing pathway in dopaminergic neurons. Consistently, we report that TOR activity in dopaminergic neurons modulates sleep/wake states. Altogether, this study provides evidence that LAT1-mediated amino acid transport in dopaminergic neurons is playing a significant role in sleep/wake regulation and is providing several entry points to elucidate the role of nutrients such as amino acids in sleep/wake regulation.

L-Citrulline restores nitric oxide level and cellular uptake at the brain capillary endothelial cell line (TR-BBB cells) with glutamate cytotoxicity

ObjectiveGlutamate excitotoxicity provokes neuronal cell damage and death, leading to collapse of the blood-brain barrier (BBB). Recently, it has been reported that l-citrulline, a neutral amino acid and a major precursor of l-arginine in the nitric oxide (NO) cycle, can prevent both neuronal cell death and cerebrovascular cell loss in brain ischemia. Therefore, the objective of this study was to investigate the effect of l-citrulline on glutamate cytotoxicity in the BBB using the conditionally immortalized rat brain capillary endothelial cell line (TR-BBB cells) as an in vitro model of the BBB. MethodsCell viability was determined using MTT assay. Cellular uptake of [14C] l-citrulline and expression levels of rat large neutral amino acid transporter 1 (rLAT1), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) at mRNA level were performed using quantitative real-time polymerase chain reaction (PCR) analysis. NO production from TR-BBB cells was measured using Griess reagents. All experiments were performed after treatment of TR-BBB cells with glutamate alone or co-treatment with l-citrulline, l-arginine, and/or taurine for 24 h. Resultsl-Citrulline treatment increased cell viability, [14C] l-citrulline uptake, and the mRNA levels of LAT1 and eNOS in TR-BBB cells treated with glutamate. However, iNOS mRNA expression was inhibited by l-citrulline. NO production and transcript level of iNOS were markedly increased by glutamate treatment alone. However, co-treatment with l-citrulline, taurine, or both l-citrulline and taurine decreased NO levels and mRNA levels of iNOS in TR-BBB cells treated with glutamate. In co-treatment of TR-BBB cells with l-arginine, a NO donor, and glutamate, NO levels were increased and expression levels of iNOS mRNA were similar compared to those in cells treated with glutamate alone. Conclusionl-Citrulline can restore NO level and its cellular uptake in TR-BBB cells with glutamate cytotoxicity. Supplying l-citrulline at the BBB may provide neuroprotective effect to improve cerebrovascular dysfunction such as a brain ischemia.