Concentrations Of Neutral Amino Acids Research Articles

Involvement of ACE2 in the intestinal transport of amino acids: Possible health and nutritional consequences in altered expression

Background:Absorption of neutral amino acids across the intestinal cells is mediated by the broad neutral amino acid transporter B0AT1. Intestinal expression of B0AT1 depends on the coexistence with the membrane-attached angiotensin-converting enzyme 2 (ACE2) which binds to the B0AT1 or the sodium-dependent imino-acid transporter 1 (SIT1) in the intestinal epithelial cells. Objectives:The objective of this article is to provide an overview of the involvement of ACE2 in the transport of amino acids from the intestinal lumen and possible harmful effects on health and nutrition due to altered ACE2 expression. Methods: PubMed, Scopus, and Google Scholar were searched using the keywords ACE2 paired with intestinal and gut B0AT1, SIT1, amino acid transport or absorption, health, and nutrition. Expression of B0AT1 in the intestine was completely absent in ACE2 knockout mice. Lack of ACE2 and B0AT1 expression lead to low plasma and muscle tryptophan, niacin deficiency, decreased intestinal antimicrobial peptide, and increased susceptibility to inflammatory bowel disease in animal models. Results: Expression of ACE2 in the small intestine and its association with amino acid transporters play a crucial role in the absorption of amino acids, maintenance of structural and functional gut integrity and overall health and nutrition status of an individual. Bangladesh Journal of Medical Science Vol. 22 No. 04 October’23 Page : 729-733

Aristolochic acid-induced nephropathy is attenuated in mice lacking the neutral amino acid transporter B0AT1 (Slc6a19).

B0AT1 (Slc6a19) mediates absorption of neutral amino acids in the small intestine and in the kidneys, where it is primarily expressed in early proximal tubules (S1-S2). To determine the role of B0AT1 in nephropathy induced by aristolochic acid (AA), which targets the proximal tubule, littermate female B0AT1-deficient (Slc6a19-/-), heterozygous (Slc6a19+/-), and wild-type (WT) mice were administered AA (10 mg/kg ip) or vehicle every 3 days for 3 wk, and analyses were performed after the last injection or 3 wk later. Vehicle-treated mice lacking Slc6a19 showed normal body and kidney weight and plasma creatinine versus WT mice. The urinary glucose-to-creatinine ratio (UGCR) and urinary albumin-to-creatinine ratio (UACR) were two to four times higher in vehicle-treated Slc6a19-/- versus WT mice, associated with lesser expression of early proximal transporters Na+-glucose cotransporter 2 and megalin, respectively. AA caused tubular injury independently of B0AT1, including robust increases in cortical mRNA expression of p53, p21, and hepatitis A virus cellular receptor 1 (Havcr1), downregulation of related proximal tubule amino acid transporters B0AT2 (Slc6a15), B0AT3 (Slc6a18), and Slc7a9, and modest histological tubular damage and a rise in plasma creatinine. Absence of B0AT1, however, attenuated AA-induced cortical upregulation of mRNA markers of senescence (p16), inflammation [lipocalin 2 (Lcn2), C-C motif chemokine ligand 2 (Ccl2), and C-C motif chemokine receptor 2 (Ccr2)], and fibrosis [tissue inhibitor of metallopeptidase 1 (Timp1), transforming growth factor-β1 (Tgfb1), and collagen type I-α1 (Col1a1)], associated with lesser fibrosis staining, lesser suppression of proximal tubular organic anion transporter 1, restoration of Na+-glucose cotransporter 2 expression, and prevention of the AA-induced fivefold increase in the urinary albumin-to-creatinine ratio observed in WT mice. The data suggest that proximal tubular B0AT1 is important for the physiology of renal glucose and albumin retention but potentially deleterious for the kidney response following AA-induced kidney injury.NEW & NOTEWORTHY Based on insights from studies manipulating glucose transport, the hypothesis has been proposed that inhibiting intestinal uptake or renal reabsorption of energy substrates has unique therapeutic potential to improve metabolic disease and kidney outcome in response to injury. The present study takes this idea to B0AT1, the major transporter for neutral amino acids in the intestine and kidney, and shows that its absence attenuates aristolochic acid-induced nephropathy.

Effect of Varying Levels of Dietary Tryptophan on Aggression and Abnormal Behavior in Growing Pigs.

Body lesions, resulting from tail-biting and ear-biting, can result in decreased health and welfare in pigs. Tryptophan, an indispensable amino acid, is needed to support protein deposition, and the synthesis of the neurotransmitter serotonin, which is important to mood, sleep-wake and eating patterns and might play a role in aggression and abnormal behavior. Two randomized block design studies were conducted to assess the influence of varying dietary tryptophan levels on aggression and abnormal behavior in 8-week-old pigs. Six diets were formulated which met or exceeded all nutrient requirements yet differed according to the dietary tryptophan content. The first study included control (100% standardized ileal digestible tryptophan), supplemented (175% standardized ileal digestible tryptophan), and supplement-plus (250% standardized ileal digestible tryptophan) experimental diets, while the second study included deficient (80% standardized ileal digestible tryptophan), adequate control (105% standardized ileal digestible tryptophan), and extra-tryptophan (130% standardized ileal digestible tryptophan) experimental diets. Concentrations of plasma tryptophan and large neutral amino acids (tyrosine, isoleucine, leucine, valine, and phenylalanine) were analyzed using ultra-performance liquid chromatography and the tryptophan to large neutral amino acid ratio was calculated. Analysis for time active, lying, and engaging in aggressive interactions was carried out using 10-min scan samples to determine behavioral time budgets of the pigs on different experimental diets. Pigs fed diets with supplemented tryptophan had higher concentrations of both plasma tryptophan and tryptophan to large neutral amino acid ratio compared to the pigs fed the control diet (P < 0.05) in the first study, while no significant differences were detected for plasma tryptophan or the tryptophan to large neutral amino acid ratio in the second study. Diet did not have an effect (P > 0.05) on weight, feed intake or behavior throughout the studies. The results suggest that an increase in dietary tryptophan relative to large neutral amino acids, fed for 29 days, impacts circulating plasma tryptophan and therefore, serotonin concentrations in the pig. Despite an increase in circulating plasma tryptophan in response to an increase in dietary tryptophan in the first study, we failed to see an impact of the dietary treatment on body, tail and ear-biting behavior under the conditions studied.

Phenylketonuria oxidative stress and energy dysregulation: Emerging pathophysiological elements provide interventional opportunity.

Phenylalanine hydroxylase (PAH) deficient phenylketonuria (PKU) is rightfully considered the paradigm treatable metabolic disease. Dietary substrate restriction (i.e. phenylalanine (Phe) restriction) was applied >60 years ago and remains the primary PKU management means. The traditional model of PKU neuropathophysiology dictates blood Phe over-representation directs asymmetric blood:brain barrier amino acid transport through the LAT1 transporter with subsequent increased cerebral Phe concentration and low concentrations of tyrosine (Tyr), tryptophan (Trp), leucine (Leu), valine (Val), and isoleucine (Ile). Low Tyr and Trp concentrations generate secondary serotonergic and dopaminergic neurotransmitter paucities, widely attributed as drivers of PKU neurologic phenotypes. White matter disease, a central PKU characteristic, is ascribed to Phe-mediated tissue toxicity. Impaired cerebral protein synthesis, by reduced concentrations of non-Phe large neutral amino acids, is another cited pathological mechanism. The PKU amino acid transport model suggests Phe management should be more efficacious than is realized, as even early identified, continuously treated patients that retain therapy compliance into adulthood, demonstrate neurologic disease elements. Reduced cerebral metabolism was an early-recognized element of PKU pathology. Legacy data (late 1960's to mid-1970's) determined the Phe catabolite phenylpyruvate inhibits mitochondrial pyruvate transport. Respirometry of Pahenu2 cerebral mitochondria have attenuated respiratory chain complex 1 induction in response to pyruvate substrate, indicating reduced energy metabolism. Oxidative stress is intrinsic to PKU and Pahenu2 brain tissue presents increased reactive oxygen species. Phenylpyruvate inhibits glucose-6-phosphate dehydrogenase that generates reduced niacinamide adenine dinucleotide phosphate the obligatory cofactor of glutathione reductase. Pahenu2 brain tissue metabolomics identified increased oxidized glutathione and glutathione disulfide. Over-represented glutathione disulfide argues for reduced glutathione reductase activity secondary to reduced NADPH. Herein, we review evidence of energy and oxidative stress involvement in PKU pathology. Data suggests energy deficit and oxidative stress are features of PKU pathophysiology, providing intervention-amenable therapeutic targets to ameliorate disease elements refractory to standard of care.

Abiotic Aggregation of Organic Matter in Coastal and Estuarine Waters: Cases in the Eastern Long Island Sound, USA

Organic aggregates, which formed from small particles and dissolved material, were chemically characterized in the Long Island Sound coastal waters. In this study, six aggregation experiments were conducted on low-salinity samples (the Thames River, CT, USA; salinity of 6.3–6.8) and high-salinity samples (the coast of Avery Point, CT, USA; salinity of 21.4–26.7). Water samples were incubated on a roller table for two days under dark conditions to generate laboratory-made aggregates. Particulate organic carbon (POC) concentrations increased 5–39% after two days of rolling. A higher POC increase occurred in low-salinity samples. The concentrations of neutral aldoses and amino acids, as well as their C- and N-yields, decreased during the experiments (except for particulate hydrolysable amino acid in low-salinity samples), while bacterial abundance increased 50–476%, indicating microbial degradation of biologically labile organic matter. Particulate hydrolysable amino acid was preferentially preserved in P-limited systems. An enrichment factor analysis showed the preferential microbial degradation of particulate hydrolysable neutral aldose and glucose appeared as the most labile aldose. The increase in bulk POC and the decrease in the fraction of labile organic carbon (neutral aldose and amino acid) in the particulate phase resulted in an accumulation of uncharacterized (presumably more refractory) particulate organic matter.

In vivo antidiabetic activity of nimesulide due to inhibition of amino acid transport

AbstractInhibiting the intestinal and renal neutral amino acid transporter B0AT1 by genetic means has improved insulin sensitivity in mice, but there are no antagonists available for preclinical or clinical use. Since the anti‐inflammatory agent nimesulide selectively inhibited B0AT1 in vitro, we hypothesized that nimesulide exhibits in vivo potential to restrict neutral amino acid absorption and, therefore, may improve insulin sensitivity. The dose‐related effect of nimesulide (10 to 100 mg/kg, PO) on intestinal absorption of neutral amino acids was estimated in C57 mice. The effect of nimesulide (50 mg/kg, PO) on renal resorption of amino acids was also assessed. The effect of chronic nimesulide (50 mg/kg, PO, BID for 14 days) was assessed in high protein diet‐fed C57 mice, diet‐induced obese mice and obese and diabetic db/db mice. Acute and chronic nimesulide treatment decreased absorption of neutral amino acids and increased their urinary excretion. Nimesulide treatment improved insulin sensitivity and glycemic control, increased GLP‐1, decreased liver lipids and improved FGF‐21 in serum. Nimesulide improved insulin sensitivity and glucose tolerance by inhibiting neutral amino acid transport in the intestine and kidneys. Thus, it can serve as a tool compound for in vivo B0AT1 inhibition.

Molecular Insights into SARS-CoV2-Induced Alterations of the Gut/Brain Axis.

For a yet unknown reason, a substantial share of patients suffering from COVID-19 develop long-lasting neuropsychiatric symptoms ranging from cognitive deficits to mood disorders and/or an extreme fatigue. We previously reported that in non-neural cells, angiotensin-1 converting enzyme 2 (ACE2), the gene coding for the SARS-CoV2 host receptor, harbors tight co-expression links with dopa-decarboxylase (DDC), an enzyme involved in the metabolism of dopamine. Here, we mined and integrated data from distinct human expression atlases and found that, among a wide range of tissues and cells, enterocytes of the small intestine express the highest expression levels of ACE2, DDC and several key genes supporting the metabolism of neurotransmitters. Based on these results, we performed co-expression analyses on a recently published set of RNA-seq data obtained from SARS-CoV2-infected human intestinal organoids. We observed that in SARS-CoV2-infected enterocytes, ACE2 co-regulates not only with DDC but also with a specific group of genes involved in (i) the dopamine/trace amines metabolic pathway, (ii) the absorption of microbiota-derived L-DOPA and (iii) the absorption of neutral amino acids serving as precursors to neurotransmitters. We conclude that in patients with long COVID, a chronic infection and inflammation of small intestine enterocytes might be indirectly responsible for prolonged brain alterations.

The Amino Acid Transporter OsAAP4 Contributes to Rice Tillering and Grain Yield by Regulating Neutral Amino Acid Allocation through Two Splicing Variants

BackgroundAmino acids, which are transported by amino acid transporters, are the major forms of organic nitrogen utilized by higher plants. Among the 19 Amino Acid Permease transporters (AAPs) in rice, only a small number of these genes have been reported to influence rice growth and development. However, whether other OsAAPs are responsible for rice growth and development is unclear.ResultsIn this study, we demonstrate that OsAAP4 promoter sequences are divergent between Indica and Japonica, with higher expression in the former, which produces more tillers and higher grain yield than does Japonica. Overexpression of two different splicing variants of OsAAP4 in Japonica ZH11 significantly increased rice tillering and grain yield as result of enhancing the neutral amino acid concentrations of Val, Pro, Thr and Leu. OsAAP4 RNA interference (RNAi) and mutant lines displayed opposite trends compared with overexpresing (OE) lines. In addition, exogenous Val or Pro at 0.5 mM significantly promoted the bud outgrowth of lines overexpressing an OsAAP4a splicing variant compared with ZH11, and exogenous Val or Pro at 2.0 mM significantly enhanced the bud outgrowth of lines overexpressing splicing variant OsAAP4b compared with ZH11. Of note, the results of a protoplast amino acid-uptake assay showed that Val or Pro at different concentrations was specifically transported and accumulated in these overexpressing lines. Transcriptome analysis further demonstrated that OsAAP4 may affect nitrogen transport and metabolism, and auxin, cytokinin signaling in regulating rice tillering.ConclusionOur results suggested that OsAAP4 contributes to rice tiller and grain yield by regulating neutral amino acid allocation through two different splicing variants and that OsAAP4 might have potential applications in rice breeding.

The Differential Metabolic Profiles Between Deltamethrin-Resistant and -Susceptible Strains of Aedes albopictus (Diptera: Culicidae) by 1H-NMR.

Metabolomics can indicate the physiological and biochemical responses of mosquitoes to different stimulants, including insecticides, which allow them to adapt to different inhospitable environments. Though metabolic differences between insecticide-resistant and -susceptible strains have been established for other mosquito species, such as Anopheles and Culex, it is yet to be done for Aedes albopictus (Skuse). In this study, nuclear magnetic resonance (NMR) spectroscopy-based metabolomic analysis performed on Ae. albopictus deltamethrin-resistant and -susceptible strains showed significant differences in amino acid, organic acid, and sugar metabolism. Concentrations of neutral amino acids and sugars tended to be lower in the deltamethrin-resistant strain than in the deltamethrin-suceptible strain, but the concentration of basic and acidic amino acids and organic acids increased. All these changes might accommodate biochemical and physiological needs in deltamethrin-resistant mosquitoes, such as enzyme synthesis and detoxification. This was further confirmed by the predictable draft metabolic map. This is the first report using NMR spectroscopy to investigate the metabolic differences between deltamethrin-resistant and -susceptible strains of Ae. albopictus. To a certain degree, this demonstrates how Ae. albopictus develop insecticide resistance by metabolic reprograming to survive under the insecticide pressure.

Loss of the psychiatric risk factor SLC6A15 is associated with increased metabolic functions in primary hippocampal neurons.

Major depressive disorder (MDD) is one of the most severe global health problems with millions of people affected, however, the mechanisms underlying this disorder is still poorly understood. Genome-wide association studies have highlighted a link between the neutral amino acid transporter SLC6A15 and MDD. Additionally, a number of preclinical studies support the function of this transporter in modulating levels of brain neurotransmitters, stress system regulation and behavioural phenotypes related to MDD. However, the molecular and functional mechanisms involved in this interaction are still unresolved. Therefore, to investigate the effects of the SLC6A15 transporter, we used hippocampal tissue from Slc6a15-KO and wild-type mice, together with several in-vitro assays in primary hippocampal neurons. Utilizing a proteomics approach we identified differentially regulated proteins that formed a regulatory network and pathway analysis indicated significantly affected cellular domains, including metabolic, mitochondrial and structural functions. Furthermore, we observed reduced release probability at glutamatergic synapses, increased mitochondrial function, higher GSH/GSSG redox ratio and an improved neurite outgrowth in primary neurons lacking SLC6A15. In summary, we hypothesize that by controlling the intracellular concentrations of neutral amino acids, SLC6A15 affects mitochondrial activity, which could lead to alterations in neuronal structure and activity. These data provide further indication that a pharmacological or genetic reduction of SLC6A15 activity may indeed be a promising approach for antidepressant therapy.

COVID-19 and Hartnup disease: an affair of intestinal amino acid malabsorption

Since the outbreak of COVID-19, clinicians have tried every effort to fight the disease, and multiple drugs have been proposed. However, no proven effective therapies currently exist, and different clinical phenotypes complicate the situation. In clinical practice, many severe or critically ill COVID-19 patients developed gastrointestinal (GI) disturbances, including vomiting, diarrhoea, or abdominal pain, even in the absence of cough and dyspnea. Understanding the mechanism of GI disturbances is warranted for exploring better clinical care for COVID-19 patients. With evidence collected from clinical studies on COVID-19 and basic research on a rare genetic disease (i.e., Hartnup disorder), we put forward a novel hypothesis to elaborate an effective nutritional therapy. We hypothesize that SARS-CoV-2 spike protein, binding to intestinal angiotensin-converting enzyme 2, negatively regulates the absorption of neutral amino acids, and this could explain not only the GI, but also systemic disturbances in COVID-19. Amino acid supplements could be recommended.Level of evidence No level of evidence: Hypothesis article.

The amino acid transporter AAP1 mediates growth and grain yield by regulating neutral amino acid uptake and reallocation in Oryza sativa.

Nitrogen (N) is a major element necessary for crop yield. In most plants, organic N is primarily transported in the form of amino acids. Here, we show that amino acid permease 1 (AAP1) functions as a positive regulator of growth and grain yield in rice. We found that the OsAAP1 gene is highly expressed in rice axillary buds, leaves, and young panicles, and that the OsAAP1 protein is localized to both the plasma membrane and the nuclear membrane. Compared with the wild-type ZH11, OsAAP1 overexpression (OE) lines exhibited increased filled grain numbers as a result of enhanced tillering, while RNAi and CRISPR (clustered regularly interspaced short palindromic repeat; Osaap1) knockout lines showed the opposite phenotype. In addition, OsAAP1-OE lines had higher concentrations of neutral and acidic amino acids, but lower concentrations of basic amino acids in the straw. An exogenous treatment with neutral amino acids promoted axillary bud outgrowth more strongly in the OE lines than in the WT, RNAi, or Osaap1 lines. Transcriptome analysis of Osaap1 further demonstrated that OsAAP1 may affect N transport and metabolism, and auxin, cytokinin, and strigolactone signaling in regulating rice tillering. Taken together, these results support that increasing neutral amino acid uptake and reallocation via OsAAP1 could improve growth and grain yield in rice.

Antioxidant activity and duodenum transporter gene expression in quail fed citric acid

The study aimed to evaluate the effects of citric acid supplementation on the performance and the gene expression in the duodenum of Japanese quail. Genes related to antioxidant activity: superoxide dismutase ( SOD ) and glutathione peroxidase 7 ( GPX7 ); and genes related to nutrient uptake in the enterocytes’ apical membrane: sodium-glucose cotransporter 1 ( SGLT1 , responsible for the glucose absorption), glucose transporter 2 ( GLUT2 ) and B 0 AT1 (responsible for the absorption of neutral amino acids in brush board membrane) were evaluated. For this, 270 laying quails ( Coturnix japonica ) were fed a diet supplemented with 0%, 0.6% or 1.2% of citric acid. Gene expression was evaluated by the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) at 35 days old (n = 5). The highest expression of the SGLT1 gene was observed in quails fed the 1.2% citric acid supplemented diet (0.120 AU). Birds fed diets with both levels of citric acid supplementation showed a higher B 0 AT1 gene expression than quails fed no citric acid. Quails fed the 1.2% of citric acid supplemented diet had the lowest SOD gene expression (0.3455 AU). The birds receiving the diets supplemented with citric acid showed a lower GPX7 gene expression than the quails fed the 0% citric acid diet. These results suggests that the effect of citric acid on gene expression in the digestive tract may have contributed to the greater weight gain and lower feed intake observed in birds fed diets supplemented with citric acid. Keywords: B 0 AT1, intestinal health, organic acid, pathogenic microorganisms, SGLT1

Propagation of Plasma L-Phenylalanine Concentration Fluctuations to the Neurovascular Unit in Phenylketonuria: An in silico Study.

Phenylketonuria (PKU) is an inherited metabolic disease characterized by abnormally high concentrations of the essential amino acid L-phenylalanine (Phe) in blood plasma caused by reduced activity of phenylalanine hydroxylase (PAH). While numerous studies have shown association between high plasma Phe concentration and intellectual impairment, it is not clear whether increased Phe fluctuations also observed in PKU affect the brain as well. To investigate this, time-resolved in vivo data on Phe and competing large neutral amino acid (LNAA) concentrations in neurons are needed, but cannot be acquired readily with current methods. We have used in silico modeling as an alternative approach to characterize the interactive dynamics of Phe and competing LNAAs (CL) in the neurovascular unit (NVU). Our results suggest that plasma Phe fluctuations can propagate into the NVU cells and change there the concentration of LNAAs, with the highest magnitude of this effect observed at low frequency and high amplitude-to-mean ratio of the plasma Phe concentration fluctuations. Our model further elucidates the effect of therapeutic LNAA supplementation in PKU, showing how abnormal concentrations of Phe and CL in the NVU move thereby toward normal physiologic levels.

Spatial mRNA Expression and Response to Fasting and Refeeding of Neutral Amino Acid Transporters slc6a18 and slc6a19a in the Intestinal Epithelium of Mozambique tilapia.

The mRNA expressions of the epithelial neutral amino acid transporters slc6a18 and slc6a19a in the five segments (HL, PMC, GL, DMC, and TS) of the intestine of Mozambique tilapia, and their responses to fasting and refeeding were investigated for a better understanding of the functional and nutritional characteristics of slc6a18 and slc6a19a. Although both slc6a18 and slc6a19a were expressed mainly in the intestine, these genes showed opposing spatial distributions along the intestine. The slc6a18 was mainly expressed in the middle (GL) and posterior (DMC and TS) intestines, while slc6a19a was specifically expressed in the anterior intestine (HL and PMC). Large decreases of amino acid concentrations from the HL to GL imply that amino acids are mainly absorbed before reaching the GL, suggesting an important role of slc6a19a in the absorption. Moreover, substantial amounts of some neutral amino acids with the isoelectric point close to 6 remain in the GL. These are most likely the remaining unabsorbed amino acids or those from of amino acid antiporters which release neutral amino acids in exchange for uptake of its substrates. These amino acids were diminished in the TS, suggesting active absorption in the posterior intestine. This suggests that slc6a18 is essential to complete the absorption of neutral amino acids. At fasting, significant downregulation of slc6a19a expression was observed from the initial up to day 2 and became stable from day 4 to day 14 in the HL and PMC suggesting that slc6a19a expression reflects nutritional condition in the intestinal lumen. Refeeding stimulates slc6a19a expression, although expressions did not exceed the initial level within 3 days after refeeding. The slc6a18 expression was decreased during fasting in the GL but no significant change was observed in the DMC. Only a transient decrease was observed at day 2 in the TS. Refeeding did not stimulate slc6a18 expression. Results in this study suggest that Slc6a18 and Slc6a19 have different roles in the intestine, and that both of these contribute to establish the efficient neutral amino acid absorption system in the tilapia.

Expression and regulation of the neutral amino acid transporter B0AT1 in rat small intestine.

Absorption of neutral amino acids across the luminal membrane of intestinal enterocytes is mediated by the broad neutral amino acid transporter B0AT1 (SLC6A19). Its intestinal expression depends on co-expression of the membrane-anchored peptidase angiotensin converting enzyme 2 (ACE2) and is additionally enhanced by aminopeptidase N (CD13). We investigated in this study the expression of B0AT1 and its auxiliary peptidases as well as its transport function along the rat small intestine. Additionally, we tested its possible short- and long-term regulation by dietary proteins and amino acids. We showed by immunofluorescence that B0AT1, ACE2 and CD13 co-localize on the luminal membrane of small intestinal villi and by Western blotting that their protein expression increases in distal direction. Furthermore, we observed an elevated transport activity of the neutral amino acid L-isoleucine during the nocturnal active phase compared to the inactive one. Gastric emptying was delayed by intragastric application of an amino acid cocktail but we observed no acute dietary regulation of B0AT1 protein expression and L-isoleucine transport. Investigation of the chronic dietary regulation of B0AT1, ACE2 and CD13 by different diets revealed an increased B0AT1 protein expression under amino acid-supplemented diet in the proximal section but not in the distal one and for ACE2 protein expression a reverse localization of the effect. Dietary regulation for CD13 protein expression was not as distinct as for the two other proteins. Ring uptake experiments showed a tendency for increased L-isoleucine uptake under amino acid-supplemented diet and in vivo L-isoleucine absorption was more efficient under high protein and amino acid-supplemented diet. Additionally, plasma levels of branched-chain amino acids were elevated under high protein and amino acid diet. Taken together, our experiments did not reveal an acute amino acid-induced regulation of B0AT1 but revealed a chronic dietary adaptation mainly restricted to the proximal segment of the small intestine.

Protein-Restricted Diets for Ameliorating Motor Fluctuations in Parkinson's Disease.

Levodopa is considered to be the most effective symptomatic drug for Parkinson's disease (PD). As the disease progresses, however, the patients are likely to experience a reduced response to levodopa and develop motor fluctuations (i.e., end-of-dose wearing off and unpredictable “on-off”). Protein-rich diets and elevated plasma concentrations of large neutral amino acids have been proved to impair the therapeutic effect of levodopa by reducing its absorption and influx into the brain. On the other hand, the protein-restricted diets including low-protein diet (LPD), protein-redistribution diet (PRD) and PRD with use of low-protein products can all improve the efficacy of levodopa in patients with motor fluctuations. However, it should be noted that protein-restricted diets may also contribute to several side effects, including dyskinesia, weight loss, and malnutrition (e.g., protein and calcium insufficiency). Together, protein-restricted diets are an effective approach to improve motor fluctuations in PD patients, while long-term adherence to these diets requires monitoring for side effects.

Molecular Characterization and Dietary Regulation of the AlaSerCys Transporter 2 in Grass Carp, Ctenopharyngodon idella

AbstractAlaSerCys Transporter 2 (ASCT2), a glutamine/amino acid transporter, plays an important role in the absorption of neutral amino acids. In this study, we cloned the full‐length cDNA of ASCT2 from grass carp, Ctenopharyngodon idella, which encodes a 541‐amino‐acid protein. Phylogenetic analysis revealed that the ASCT2 sequence of grass carp clustered with the ASCT2 from Danio rerio. ASCT2 mRNA was differentially expressed in different tissues, with a gradient expression from high to low in tissues of the liver, gill, muscle, midgut, hypophysis, hindgut, kidney, heart, foregut, brain, spleen, and gonad. Meanwhile, the quantitative real‐time polymerase chain reaction results showed that 22% crude protein diets could significantly stimulate ASCT2 gene expression. In addition, ASCT2 was expressed in higher quantities in the soybean meal group than in the fishmeal group. Furthermore, 7.5 g/kg glutamine and 2.5 g/kg glutamine dipeptide remarkably improved the expression of ASCT2. These results suggested that ASCT2 expression is regulated by the dietary protein levels and sources, as well as appropriate concentration of glutamine and glutamine dipeptide additives.

Ligand Discovery for the Alanine-Serine-Cysteine Transporter (ASCT2, SLC1A5) from Homology Modeling and Virtual Screening.

The Alanine-Serine-Cysteine transporter ASCT2 (SLC1A5) is a membrane protein that transports neutral amino acids into cells in exchange for outward movement of intracellular amino acids. ASCT2 is highly expressed in peripheral tissues such as the lung and intestines where it contributes to the homeostasis of intracellular concentrations of neutral amino acids. ASCT2 also plays an important role in the development of a variety of cancers such as melanoma by transporting amino acid nutrients such as glutamine into the proliferating tumors. Therefore, ASCT2 is a key drug target with potentially great pharmacological importance. Here, we identify seven ASCT2 ligands by computational modeling and experimental testing. In particular, we construct homology models based on crystallographic structures of the aspartate transporter GltPh in two different conformations. Optimization of the models’ binding sites for protein-ligand complementarity reveals new putative pockets that can be targeted via structure-based drug design. Virtual screening of drugs, metabolites, fragments-like, and lead-like molecules from the ZINC database, followed by experimental testing of 14 top hits with functional measurements using electrophysiological methods reveals seven ligands, including five activators and two inhibitors. For example, aminooxetane-3-carboxylate is a more efficient activator than any other known ASCT2 natural or unnatural substrate. Furthermore, two of the hits inhibited ASCT2 mediated glutamine uptake and proliferation of a melanoma cancer cell line. Our results improve our understanding of how substrate specificity is determined in amino acid transporters, as well as provide novel scaffolds for developing chemical tools targeting ASCT2, an emerging therapeutic target for cancer and neurological disorders.

Ligand Discovery for the Alanine-Serine-Cysteine Transporter (ASCT2, SLC1A5) from Homology Modeling and Virtual Screening

The Alanine-Serine-Cysteine transporter ASCT2 (SLC1A5) is a membrane protein that moves neutral amino acids into cells in exchange for outward movement of intracellular amino acid. ASCT2 is highly expressed in peripheral tissues such as the lung, kidney, intestines, and testis where it contributes to the homeostasis of intracellular concentrations of neutral amino acids. ASCT2 also plays an important role in the development of a variety of cancers such as melanoma, by transporting amino acid nutrients such as glutamine into the proliferating tumors. Therefore, ASCT2 is a key drug target with potentially great pharmacological importance. Here, we identify seven ASCT2 ligands by computational modeling and experimental testing. In particular, we construct homology models based on crystallographic structures of the aspartate transporter GltPh in two different conformations, and optimize the models’ binding sites for protein-ligand complementarity. Virtual screening of 594,166 compounds including drugs, metabolites, and fragment-like molecules from the ZINC database, followed by experimental testing of 11 top hits with functional measurements using electrophysiological methods reveals seven ligands including five activators and two inhibitors. For example, aminooxetane-3-carboxylate is a more efficient activator than any other known ASCT2 natural or unnatural substrate. Furthermore, two of the hits inhibited ASCT2 mediated glutamine uptake and proliferation of a melanoma cancer cell line. Our results improve our understanding on how substrate specificity is determined in amino acid transporters as well as provide novel scaffolds for developing drugs targeting ASCT2, an emerging therapeutic target for cancer and neurological disorders.