l-Type amino acid transporter 1 in hypothalamic neurons in mice maintains energy and bone homeostasis.

Hypothalamic neurons regulate body homeostasis by sensing and integrating changes in the levels of key hormones and primary nutrients (amino acids, glucose, and lipids). However, the molecular mechanisms that enable hypothalamic neurons to detect primary nutrients remain elusive. Here, we identified l-type amino acid transporter 1 (LAT1) in hypothalamic leptin receptor-expressing (LepR-expressing) neurons as being important for systemic energy and bone homeostasis. We observed LAT1-dependent amino acid uptake in the hypothalamus, which was compromised in a mouse model of obesity and diabetes. Mice lacking LAT1 (encoded by solute carrier transporter 7a5, Slc7a5) in LepR-expressing neurons exhibited obesity-related phenotypes and higher bone mass. Slc7a5 deficiency caused sympathetic dysfunction and leptin insensitivity in LepR-expressing neurons before obesity onset. Importantly, restoring Slc7a5 expression selectively in LepR-expressing ventromedial hypothalamus neurons rescued energy and bone homeostasis in mice deficient for Slc7a5 in LepR-expressing cells. Mechanistic target of rapamycin complex-1 (mTORC1) was found to be a crucial mediator of LAT1-dependent regulation of energy and bone homeostasis. These results suggest that the LAT1/mTORC1 axis in LepR-expressing neurons controls energy and bone homeostasis by fine-tuning sympathetic outflow, thus providing in vivo evidence of the implications of amino acid sensing by hypothalamic neurons in body homeostasis.

Effects of hydrogen peroxide and l-tryptophan on antioxidative potential, apoptosis, and mammalian target of rapamycin signaling in bovine intestinal epithelial cells.

Amino acids are primarily absorbed in the ruminant small intestine, and the small intestine is a target organ prone to oxidative stress, causing intestinal disfunction. Previous study suggested that l-Trp could benefit intestinal function and production performance. This study aimed to explore the effects of l-Trp on hydrogen peroxide (H2O2)-induced oxidative injury in bovine intestinal epithelial cells (BIEC) and the potential mechanism. The effects of l-Trp on cell apoptosis, antioxidative capacity, AA transporters, and the mammalian target of rapamycin (mTOR) signaling pathway were evaluated in BIEC treated with 0.8 mMl-Trp for 2 hours combined with or without H2O2 induction. In addition, to explore whether the effects of 0.8 mMl-Trp on oxidative stress were related to mTOR, an mTOR-specific inhibitor was used. The percentage of apoptosis was measured using flow cytometry. The relative gene abundance and protein expression in BIEC were determined using real-time PCR and Western blot assay, respectively. Results showed l-Trp at 0.4 and 0.8 mM enhanced the cell viability, and it was inhibited by l-Trp at 6.4 mM. l-Tryptophan at 0.4, 0.8, and 1.6 mM remarkably decreased the percentage of apoptosis and enhanced antioxidative capacity in H2O2-mediated BIEC. Moreover, l-Trp at 0.8 mM increased the relative gene abundance and protein expression of antioxidative enzymes and AA transporters, and the mTOR signaling pathway. The mTOR inhibitor lowered the protein expression of large neutral amino acid transporter 1, but the inhibition of mTOR did not alter the activities of catalase and superoxide dismutase or protein expression of alanine-serine-cysteine transporter 2 with or without H2O2 induction. l-Tryptophan increased catalase and superoxide dismutase activities in H2O2-mediated BIEC, although not with a present mTOR inhibitor. l-Tryptophan increased the protein expression of large neutral amino acid transporter 1 and alanine-serine-cysteine transporter 2 in H2O2-mediated BIEC with or without the presence of an mTOR inhibitor. The present work suggested that l-Trp supplementation could alleviate oxidative injury in BIEC by promoting antioxidative capacity and inhibiting apoptosis, and the mTOR signal played vital roles in the alleviation.

Leucine Supplementation in Middle-Aged Male Mice Improved Aging-Induced Vascular Remodeling and Dysfunction via Activating the Sirt1-Foxo1 Axis.

Vascular aging is associated with metabolic remodeling, and most studies focused on fatty acid and glucose metabolism. Based on our metabolomic data, leucine was significantly reduced in the aortas of aged mice. Whether leucine supplementation can reverse aging-induced vascular remodeling remains unknown. To investigate the effectiveness of leucine, male mice at 15 or 18 months were supplemented with leucine (1.5%) for 3 months. All the aged mice, with or without leucine, were sacrificed at 21 months. Blood pressure and vascular relaxation were measured. H&E, Masson's trichrome, and Elastica van Gieson staining were used to assess aortic morphology. Vascular inflammation, reactive oxidative stress (ROS), and vascular smooth muscle cell (VSMC) phenotype were also measured in mouse aortas. Compared with the 21-month-old mice without leucine, leucine supplementation from 15 months significantly improved vascular relaxation, maintained the contractile phenotype of VSMCs, and repressed vascular inflammation and ROS levels. These benefits were not observed in the mice supplemented with leucine starting from 18 months, which was likely due to the reduction in leucine transporters Slc3a2 or Slc7a5 at 18 months. Furthermore, we found benefits from leucine via activating the Sirt1-induced Foxo1 deacetylation. Our findings indicated that leucine supplementation in middle-aged mice improved aging-induced vascular remodeling and dysfunction.

High L-Type Amino Acid Transporter 1 Levels Are Associated with Chemotherapeutic Resistance in Gastric Cancer Patients.

INTRODUCTION: We investigated whether the expression of L-type amino acid transporter 1 (LAT-1) in clinical gastric cancer (GC) patients could predict patient therapeutic response to postoperative adjuvant chemotherapy. METHODS: Immunohistochemistry was used to investigate LAT-1, CD98, and phosphorylated-mammalian target of rapamycin (p-mTOR) expression in 111 GC patients. To clarify whether LAT-1 influences the therapeutic effects of chemotherapy, the correlation between disease-free survival rates and LAT-1 was determined in 2 groups: 59 patients who did not undergo postoperative adjuvant chemotherapy and 52 patients who did undergo postoperative adjuvant chemotherapy. RESULTS: LAT-1 was significantly correlated with CD98 and p-mTOR expressions. We did not find any statistically significant correlation between LAT-1 and recurrence in the nontreated group. In contrast, a significant association was found between LAT-1 expression and disease-free survival in the chemotherapy group. Moreover, multivariate regression analysis demonstrated that LAT-1 was an independent predictor of disease-free survival in the postoperative adjuvant chemotherapy group (p = 0.012). CONCLUSION: Our findings demonstrate that LAT-1 is a useful predictive marker for a successful postoperative adjuvant chemotherapy treatment.

Fetal sex modulates placental microRNA expression, potential microRNA-mRNA interactions, and levels of amino acid transporter expression and substrates: INFAT study subpopulation analysis of n-3 LCPUFA intervention during pregnancy and associations with offspring body composition.

BACKGROUND: Previously, we revealed sexually dimorphic mRNA expression and responsiveness to maternal dietary supplementation with n-3 long-chain polyunsaturated fatty acids (LCPUFA) in placentas from a defined INFAT study subpopulation. Here, we extended these analyses and explored the respective placental microRNA expression, putative microRNA-mRNA interactions, and downstream target processes as well as their associations with INFAT offspring body composition. RESULTS: We performed explorative placental microRNA profiling, predicted microRNA-mRNA interactions by bioinformatics, validated placental target microRNAs and their putative targets by RT-qPCR and western blotting, and measured amino acid levels in maternal and offspring cord blood plasma and placenta. microRNA, mRNA, protein, and amino acid levels were associated with each other and with offspring body composition from birth to 5 years of age. Forty-six differentially regulated microRNAs were found. Validations identified differential expression for microRNA-99a (miR-99a) and its predicted target genes mTOR, SLC7A5, encoding L-type amino acid transporter 1 (LAT1), and SLC6A6, encoding taurine transporter (TauT), and their prevailing significant sexually dimorphic regulation. Target mRNA levels were mostly higher in placentas from control male than from female offspring, whereas respective n-3 LCPUFA responsive target upregulation was predominantly found in female placentas, explaining the rather balanced expression levels between the sexes present only in the intervention group. LAT1 and TauT substrates tryptophan and taurine, respectively, were significantly altered in both maternal plasma at 32 weeks' gestation and cord plasma following intervention, but not in the placenta. Several significant associations were observed for miR-99a, mTOR mRNA, SLC7A5 mRNA, and taurine and tryptophan in maternal and cord plasma with offspring body composition at birth, 1 year, 3 and 5 years of age. CONCLUSIONS: Our data suggest that the analyzed targets may be part of a sexually dimorphic molecular regulatory network in the placenta, possibly modulating gene expression per se and/or counteracting n-3 LCPUFA responsive changes, and thereby stabilizing respective placental and fetal amino acid levels. Our data propose placental miR-99, SLC7A5 mRNA, and taurine and tryptophan levels in maternal and fetal plasma as potentially predictive biomarkers for offspring body composition.

Co-Expression Effect of SLC7A5/SLC3A2 to Predict Response to Endocrine Therapy in Oestrogen-Receptor-Positive Breast Cancer.

The majority of breast cancers are oestrogen-receptor-positive (ER+) and are subject to endocrine therapy; however, an unpredictable subgroup of patients will develop resistance to endocrine therapy. The SLC7A5/SLC3A2 complex is a major route for the transport of large neutral essential amino acids through the plasma membrane. Alterations in the expression and function of those amino-acid transporters lead to metabolic reprogramming, which contributes to the tumorigenesis and drug resistance. This study aims to assess the effects and roles of SLC7A5/SLC3A2 co-expression in predicting responses to endocrine therapy in patients with ER+ breast cancer. The biological and clinical impact of SLC7A5/SLC3A2 co-expression was assessed in large annotated cohorts of ER+/HER2- breast cancer with long-term follow-up at the mRNA and protein levels. In vitro experiments were conducted to investigate the effect of SLC7A5/SLC3A2 knockdown in the proliferation of cancer cells and to the sensitivity to tamoxifen. We found that proliferation-related genes are highly expressed in a subgroup of patients with high SLC7A5/SLC3A2, and knockdown of SLC7A5/SLC3A2 decreased proliferation of ER+ breast cancer cells. In patients treated with endocrine therapy, high SLC7A5/SLC3A2 co-expression was associated with poor patient outcome, and depletion of SLC7A5/SLC3A2 using siRNA increased the sensitivity of breast cancer cells to tamoxifen. On the basis of our findings, SLC7A5/SLC3A2 co-expression has the potential of identifying a subgroup of ER+/HER2- breast cancer patients who fail to benefit from endocrine therapy and could guide the choice of other alternative therapies.

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.

Piperine metabolically regulates peritoneal resident macrophages to potentiate their functions against bacterial infection.

Pepper, a daily-used seasoning for promoting appetite, is widely used in folk medicine for treating gastrointestinal diseases. Piperine is the major alkaloid in pepper and possesses a wide range of pharmacological activities. However, the mechanism for linking metabolic and medicinal activities of piperine remains unknown. Here we report that piperine robustly boosts mTORC1 activity by recruiting more system L1 amino acid transporter (SLC7A5/SLC3A2) to the cell membrane, thus promoting amino acid metabolism. Piperine-induced increase of mTORC1 activity in resident peritoneal macrophages (pMΦs) is correlated with enhanced production of IL-6 and TNF-α upon LPS stimulation. Such an enhancement of cytokine production could be abrogated by inhibitors of the mTOR signaling pathway, indicating mTOR's action in this process. Moreover, piperine treatment protected resident pMΦs from bacterium-induced apoptosis and disappearance, and increased their bacterial phagocytic ability. Consequently, piperine administration conferred mice resistance against bacterial infection and even sepsis. Our data highlight that piperine has the capacity to metabolically reprogram peritoneal resident macrophages to fortify their innate functions against bacterial infection.

Development of a cell-based screening method for compounds that inhibit or are transported by large neutral amino acid transporter 1, a key transporter at the blood-brain barrier.

Large neutral amino acid transporter 1 (LAT1) transports neutral amino acids with aromatic or branched side chains as well as their derivatives or prodrugs. Because the transporter is highly expressed at the blood-brain barrier and in some tumor cells, it is a potential target to treat brain disease and cancer. Therefore, it is essential to develop a method to screen for LAT1 inhibitors or for therapeutic compounds that it can transport. In this study, one such method was developed that combines an in vitro cell-based assay with high-throughput ultra-performance liquid chromatography triple quadrupole mass spectrometry (UPLC-QQQ-MS). Using this method, candidate compounds could be tested for the ability to inhibit or to compete with uptake of gabapentin, an LAT1 substrate, in HT-29 cells, which abundantly express the transporter. Gabapentin uptake is measured by mass spectrometry, which requires as little as 6min/sample and will enable analysis of large numbers of samples. We anticipate that the method will be useful to identify LAT1 inhibitors or substrates without the need for animals or radioactive labeling.

Dietary L-arginine supplementation protects weanling pigs from deoxynivalenol-induced toxicity.

This study was conducted to determine the positive effects of dietary supplementation with L-arginine (Arg) on piglets fed a deoxynivalenol (DON)-contaminated diet. A total of eighteen, 28-day-old healthy weanling pigs were randomly assigned into one of three groups: uncontaminated basal diet (control group), 6 mg/kg DON-contaminated diet (DON group) and 6 mg/kg DON + 1% L-arginine (DON + ARG group). After 21 days of Arg supplementation, piglets in the DON and DON + ARG groups were challenged by feeding 6 mg/kg DON-contaminated diet for seven days. The results showed that DON resulted in damage to piglets. However, clinical parameters, including jejunal morphology, amino acid concentrations in the serum, jejunum and ileum, were improved by Arg (p < 0.05). Furthermore, the mRNA levels for sodium-glucose transporter-1 (SGLT-1), glucose transporter type-2 (GLUT-2) and y(+)L-type amino acid transporter-1 (y(+)LAT-1) were downregulated in the DON group, but the values were increased in the DON + ARG group (p < 0.05). Collectively, these results indicate that dietary supplementation with Arg exerts a protective role in pigs fed DON-contaminated diets.

L-amino acid transporter 1 may be a prognostic marker for local progression of prostatic cancer under expectant management.

BACKGROUND: Oncocytic L-amino acid transporter (LAT) 1 could be a target of new molecular therapy against malignancies. OBJECTIVE: To assess the correlation between overexpression of LAT1 and local progression (LP) in prostatic carcinoma (PC) patients under expectant management (EM). METHODS: This retrospective study analyzed 109 patients with PC who received EM from 1991 to 2006. The expression of LAT1, LAT2, and CD98, as well as Ki-67 labeling indices (LI), was analyzed immunohistochemically in first biopsy or TUR samples diagnosed as adenocarcinomas. RESULTS: Of the 109 patients, 44 (40%) showed LP on clinical examinations, whereas 65 showed stable disease (SD). LAT1 score and intensity were significantly higher in the LP than in the SD group, as well as among Gleason score (GS)-low (GS < 7) patients who were associated with low-risk. When the LP group was subdivided by D'Amico risk category (low-, intermediate- and high-risk groups), each showed higher LAT1 expression than the SD group. LAT1 expression did not correlate with GS or Ki-67 LI. CONCLUSIONS: Independently of GS, aberrant overexpression of LAT1 in prostatic adenocarcinoma could predict LP under EM. Although prostate biopsy samples are small, LAT1 may be a novel prognostic biomarker of LP.

Effects of dietary n-6:n-3 PUFA ratio on fatty acid composition, free amino acid profile and gene expression of transporters in finishing pigs.

Revealing the expression patterns of fatty acid and amino acid transporters as affected by dietary n-6:n-3 PUFA ratio would be useful for further clarifying the importance of the balance between n-6 and n-3 PUFA. A total of ninety-six finishing pigs were fed one of four diets with the ratio of 1:1, 2·5:1, 5:1 and 10:1. Pigs fed the dietary n-6:n-3 PUFA ratio of 5:1 had the highest (P< 0·05) daily weight gain, and those fed the dietary n-6:n-3 PUFA ratio of 1:1 had the largest loin muscle area (P< 0·01). The concentration of n-3 PUFA was raised as the ratio declined (P< 0·05) in the longissimus dorsi and subcutaneous adipose tissue. The contents of tryptophan, tasty amino acids and branched-chain amino acids in the longissimus dorsi were enhanced in pigs fed the dietary n-6:n-3 PUFA ratios of 1:1-5:1. The mRNA expression level of the fatty acid transporter fatty acid transport protein-1 (FATP-1) was declined (P< 0·05) in the longissimus dorsi of pigs fed the dietary n-6:n-3 PUFA ratios of 1:1-5:1, and increased (P< 0·05) in the subcutaneous adipose tissue of pigs fed the dietary n-6:n-3 PUFA ratios of 5:1 and 10:1. The expression profile of FATP-4 was similar to those of FATP-1 in the adipose tissue. The mRNA expression level of the amino acid transceptors LAT1 and SNAT2 was up-regulated (P< 0·05) in the longissimus dorsi of pigs fed the dietary n-6:n-3 PUFA ratios of 1:1 and 2·5:1. In conclusion, maintaining the dietary n-6:n-3 PUFA ratios of 1:1-5:1 would facilitate the absorption and utilisation of fatty acids and free amino acids, and result in improved muscle and adipose composition.

L-Leucine and L-isoleucine enhance growth of BBN-induced urothelial tumors in the rat bladder by modulating expression of amino acid transporters and tumorigenesis-associated genes.

We investigated the underlying mechanisms of L-leucine and L-isoleucine mediated promotion of bladder carcinogenesis using an initiation-promotion model. Rats were administered N-butyl-N-(4-hydroxybutyl) nitrosamine for 4 weeks and then fed AIN-93G basal diet or diet supplemented with L-leucine or L-isoleucine for 8 weeks followed by the basal diet for another 8 weeks. At the end of the experiment, week 20, there was a significant elevation of papillary and nodular (PN) hyperplasia multiplicity in the amino acid groups. L-Leucine and L-isoleucine transporters were up-regulated in PN hyperplasias and/or bladder tumors compared with concomitant normal-appearing bladder urothelium at weeks 12 and/or 20 in all groups. In addition, in normal-appearing bladder urothelium, significantly increased mRNA levels of y+LAT1, LAT2, LAT4, and 4F2hc were observed in the amino acid groups compared with the BBN control group at both weeks 12 and 20, and increased mRNA levels of LAT1 were observed at week 20. Furthermore, up-regulation of TNF-α, c-fos, ß-catenin, p53, p21(Cip1/WAF1), cdk4, cyclin D1 and caspase 3 in the amino acid groups was detected in normal-appearing bladder urothelium. Overall, our results indicate that supplementation with l-leucine or l-isoleucine enhanced growth of bladder urothelial tumors by triggering expression of amino acid transporters and tumorigenesis-associated genes.

Lysinuric protein intolerance (LPI): a multi organ disease by far more complex than a classic urea cycle disorder.

Lysinuric protein intolerance (LPI) is an inherited defect of cationic amino acid (lysine, arginine and ornithine) transport at the basolateral membrane of intestinal and renal tubular cells caused by mutations in SLC7A7 encoding the y(+)LAT1 protein. LPI has long been considered a relatively benign urea cycle disease, when appropriately treated with low-protein diet and l-citrulline supplementation. However, the severe clinical course of this disorder suggests that LPI should be regarded as a severe multisystem disease with uncertain outcome. Specifically, immune dysfunction potentially attributable to nitric oxide (NO) overproduction secondary to arginine intracellular trapping (due to defective efflux from the cell) might be a crucial pathophysiological route explaining many of LPI complications. The latter comprise severe lung disease with pulmonary alveolar proteinosis, renal disease, hemophagocytic lymphohistiocytosis with subsequent activation of macrophages, various auto-immune disorders and an incompletely characterized immune deficiency. These results have several therapeutic implications, among which lowering the l-citrulline dosage may be crucial, as excessive citrulline may worsen intracellular arginine accumulation.

Lysinuric protein intolerance: reviewing concepts on a multisystem disease.

Lysinuric protein intolerance (LPI) is an inherited aminoaciduria caused by defective cationic amino acid transport at the basolateral membrane of epithelial cells in intestine and kidney. LPI is caused by mutations in the SLC7A7 gene, which encodes the y(+)LAT-1 protein, the catalytic light chain subunit of a complex belonging to the heterodimeric amino acid transporter family. LPI was initially described in Finland, but has worldwide distribution. Typically, symptoms begin after weaning with refusal of feeding, vomiting, and consequent failure to thrive. Hepatosplenomegaly, hematological anomalies, neurological involvement, including hyperammonemic coma are recurrent clinical features. Two major complications, pulmonary alveolar proteinosis and renal disease are increasingly observed in LPI patients. There is extreme variability in the clinical presentation even within individual families, frequently leading to misdiagnosis or delayed diagnosis. This condition is diagnosed by urine amino acids, showing markedly elevated excretion of lysine and other dibasic amino acids despite low plasma levels of lysine, ornithine, and arginine. The biochemical diagnosis can be uncertain, requiring confirmation by DNA testing. So far, approximately 50 different mutations have been identified in the SLC7A7 gene in a group of 142 patients from 110 independent families. No genotype-phenotype correlation could be established. Therapy requires a low protein diet, low-dose citrulline supplementation, nitrogen-scavenging compounds to prevent hyperammonemia, lysine, and carnitine supplements. Supportive therapy is available for most complications with bronchoalveolar lavage being necessary for alveolar proteinosis.

L-type amino-acid transporter 1 expression predicts the response to preoperative hyperthermo-chemoradiotherapy for advanced rectal cancer.

AIM: To evaluate whether expression of L-type amino acid transporter 1 (LAT1) in pretreatment rectal cancer biopsies is predictive of tumour response to neoadjuvant hyperthermo-chemoradiotherapy (HCRT). PATIENTS AND METHODS: Forty-four patients with rectal adenocarcinoma who received neoadjuvant HCRT were investigated. LAT1 expression was immunohistochemically evaluated using pretreatment biopsies. The operation was performed after 2-3 months following HCRT and each resected specimen was graded by the histological criteria of the Japanese Classification of Colorectal Carcinoma. RESULTS: A positive LAT1 expression was recognized in 50.0% (22/44) of patients. Resected specimens were divided into 2 groups according to the histological grading criteria: good response (n=29) and poor response (n=15). LAT1-negative tumours had an 81.8% probability of good response and 18.2% probability of poor response. LAT1 expression showed marginally significant association with response to HCRT (p=0.05). CONCLUSION: LAT1 may be a useful predictive marker of response to HCRT in rectal cancer.

Expression of amino acid transporter LAT1 and 4F2hc in the healing process after the implantation of a tooth ash and plaster of Paris mixture.

BACKGROUND: In order to elucidate the expression pattern of L-type amino acid transporter 1 (LAT1) in the bone formation process, the expressions of LAT1 and its subunit 4F2 heavy chain (4F2hc) were investigated in the healing process after the implantation of a tooth ash-plaster of Paris mixture in rats with calvarial osseous defect. MATERIALS AND METHODS: Circular calvarial defects (8 mm in diameter) were made midparietally. The rats were divided into 2 groups, 1 control group and 1 experimental group. In the control group, the defect was only covered with a soft tissue flap (control group); in the experimental group, it was filled with a mixture of tooth ash and plaster of Paris (2:1 by weight; mixture group). The rats were sacrificed at 1, 2, 4 and 8 weeks after operation and RT-PCR and immunohistochemical analyses were performed. RESULTS: In the RT-PCR analysis, the expressions of the LAT1 and 4F2hc mRNAs were slightly stronger in the experimental group than in the control group. In the immunohistochemical analysis, at 1 week after operation, the LAT1 protein and its subunit 4F2hc protein were mainly expressed in the osteoblasts, osteocytes and interstitial tissues of the area around the defect and the inner part of newly forming bone in both groups. The expressions of LAT1 and 4F2hc proteins were decreased at 2 and 4 weeks after operation. The LAT1 and 4F2hc proteins were scarcely expressed at 8 weeks after operation in both groups. The expressions of LAT1 and 4F2hc proteins were slightly stronger in the mixture group than in the control group. CONCLUSION: These results suggest that the LAT1 and its subunit 4F2hc are highly expressed in the early stage of new bone formation and may have an important role in providing cells with neutral amino acids, including several essential amino acids, at that stage.

Traumatic axonal injury in the perisomatic domain triggers ultrarapid secondary axotomy and Wallerian degeneration.

Traumatic axonal injury (TAI) arising from diffuse brain injury (DBI) results in focally impaired axonal transport with progressive swelling and delayed disconnection over several hours within brainstem axons. Neocortical DBI-mediated perisomatic axotomy does not result in neuronal death, suggesting that a comparably delayed axotomy progression was responsible for this unanticipated response. To evaluate delayed perisomatic axotomy, the current study was initiated. Rats received intracerebroventricular 10-kDa dextran followed by moderate midline/central fluid percussion injury (FPI) or FPI alone. At 15, 30, 60, and 180 min post-injury, light and transmission electron microscopy identified impaired axonal transport via antibodies targeting amyloid precursor protein (APP), while double-label fluorescent microscopy explored concomitant focal axolemmal alterations via dextran-APP co-localization. At 15 min post-injury, perisomatic TAI was identified with LM within dorsolateral and ventral posterior thalamic nuclei. Using TEM, many sustaining somata and related proximal/distal axonal segments revealed normal ultrastructural detail that was continuous with focal axonal swellings characterized by cytoskeletal and organelle pathology. In other cases, axotomy was confirmed by loss of axonal continuity distal to the swelling. By 30 min post-injury, perisomatic axotomy predominated. By 60-180 min, somatic, proximal axonal segment, and swelling ultrastructure were comparable to earlier time points although swelling diameter increased. Distal axonal segment ultrastructure now revealed the initial stages of Wallerian degeneration. The site of perisomatic axotomy did not internalize dextran, suggesting that its pathogenesis occurred independent of altered axolemmal permeability. Collectively, this DBI-mediated ultrarapid perisomatic axotomy and its sequelae further illustrate the varied axonal responses to trauma.

Isoform selectivity of 3-125I-iodo-alpha-methyl-L-tyrosine membrane transport in human L-type amino acid transporters.

UNLABELLED: 3-(123)I-Iodo-alpha-methyl-L-tyrosine ((123)I-IMT) has been developed for SPECT of amino acid transport imaging. We examined the isoform selectivity of (125)I-IMT transport of the 2 human L-type amino acid transporters, hLAT1 and hLAT2, with human 4F2hc-coexpressed Xenopus laevis oocytes. METHODS: An uptake study of (125)I-IMT was performed using transporter-expressed X. laevis oocytes. Oocytes were injected with 17.6 ng of hLAT1 or hLAT2 complementary RNA (cRNA) and 7.4 ng of h4F2hc cRNA in a molar ratio of 1:1. Two days after injection, the uptake of (125)I-IMT was measured in the Na(+)-free uptake solution containing 18.5 kBq of noncarrier-added (125)I-IMT. After incubation for 30 min at room temperature, radioactivity of the oocytes was determined. RESULTS: Of the 2 hLAT isoforms and h4F2hc-coexpressed X. laevis oocytes, (125)I-IMT uptake via hLAT1 was 5.95-fold higher than that via hLAT2 (P < 0.005). CONCLUSION: (125)I-IMT transport was hLAT1 selective. Investigations on the isoform selectivity of (125)I-IMT transport with other transporters are anticipated.

Transport of a neurotoxicant by molecular mimicry: the methylmercury-L-cysteine complex is a substrate for human L-type large neutral amino acid transporter (LAT) 1 and LAT2.

Methylmercury (MeHg) readily crosses cell membrane barriers to reach its target tissue, the brain. Although it is generally assumed that this rapid transport is due to simple diffusion, recent studies have demonstrated that MeHg is transported as a hydrophilic complex, and possibly as an L-cysteine complex on the ubiquitous L-type large neutral amino acid transporters (LATs). To test this hypothesis, studies were carried out in Xenopus laevis oocytes expressing two of the major L-type carriers in humans, LAT1-4F2 heavy chain (4F2hc) and LAT2-4F2hc. Oocytes expressing LAT1-4F2hc or LAT2-4F2hc demonstrated enhanced uptake of [(14)C]MeHg when administered as the L-cysteine or D,L-homocysteine complexes, but not when administered as the D-cysteine, N -acetyl-L-cysteine, penicillamine or GSH complexes. Kinetic analysis of transport indicated that the apparent affinities ( K (m)) of MeHg-L-cysteine uptake by LAT1 and LAT2 (98+/-8 and 64+/-8 microM respectively) were comparable with those for methionine (99+/-9 and 161+/-11 microM), whereas the V (max) values were higher for MeHg-L-cysteine, indicating that it may be a better substrate than the endogenous amino acid. Uptake and efflux of [(3)H]methionine and [(14)C]MeHg-L-cysteine were trans -stimulated by leucine and phenylalanine, but not by glutamate, indicating that MeHg-L-cysteine is both a cis - and trans -substrate. In addition, [(3)H]methionine efflux was trans -stimulated by leucine and phenylalanine even in the presence of an inwardly directed methionine gradient, demonstrating concentrative transport by both LAT1 and LAT2. The present results describe a major molecular mechanism by which MeHg is transported across cell membranes and indicate that metal complexes may form a novel class of substrates for amino acid carriers. These transport proteins may therefore participate in metal ion homoeostasis and toxicity.