Amino Acid Response Research Articles

Old and young duplicate genes reveal different responses to environmental changes in Arachis duranensis.

Old and young duplicate genes have been reported in some organisms. However, little is known about the properties of old and young duplicate genes in Arachis. Here, we have identified old and young duplicate genes in Arachis duranensis, and analyzed the evolution, gene complexity, gene expression pattern, and functional divergence between old and young duplicate genes. Our results showed different evolutionary, gene complexity and gene expression patterns, as well as differing correlations between old and young duplicate genes. Gene ontology results showed that old duplicate genes play a crucial role in lipid and amino acid biosynthesis and the oxidation-reduction process and that young duplicate genes are preferentially involved in photosynthesis and response to biotic stimulus. Transcriptome data sets revealed that most old and young duplicate genes had asymmetric function, and only a few duplicate genes exhibited symmetric function under drought and nematode stress. We found that old duplicate genes are preferentially involved in lipid and amino acid metabolism and response to abiotic stress, while young duplicate genes are likely to participate in photosynthesis and response to biotic stress. This work provides a better understanding of the evolution and functional divergence of old and young duplicate genes in A. duranensis.

Amino acid response by Halofuginone in Cancer cells triggers autophagy through proteasome degradation of mTOR

BackgroundIn the event of amino acid starvation, the cell activates two main protective pathways: Amino Acid starvation Response (AAR), to inhibit global translation, and autophagy, to recover the essential substrates from degradation of redundant self-components. Whether and how AAR and autophagy (ATG) are cross-regulated and at which point the two regulatory pathways intersect remain unknown. Here, we provide experimental evidence that the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) specifically located at the lysosome level links the AAR with the autophagy pathway.MethodsAs an inducer of the AAR, we used halofuginone (HF), an alkaloid that binds to the prolyl-tRNA synthetase thus mimicking the unavailability of proline (PRO). Induction of AAR was determined assessing the phosphorylation of the eukaryotic translation initiation factor (eIF) 2α. Autophagy was monitored by assessing the processing and accumulation of microtubule-associated protein 1 light chain 3 isoform B (LC3B) and sequestosome-1 (p62/SQSTM1) levels. The activity of mTORC1 was monitored through assessment of the phosphorylation of mTOR, (rp)S6 and 4E-BP1. Global protein synthesis was determined by puromycin incorporation assay. mTORC1 presence on the membrane of the lysosomes was monitored by cell fractionation and mTOR expression was determined by immunoblotting.ResultsIn three different types of human cancer cells (thyroid cancer WRO cells, ovarian cancer OAW-42 cells, and breast cancer MCF-7 cells), HF induced both the AAR and the autophagy pathways time-dependently. In WRO cells, which showed the strongest induction of autophagy and of AAR, global protein synthesis was little if any affected. Consistently, 4E-BP1 and (rp)S6 were phosphorylated. Concomitantly, mTOR expression and activation declined along with its detachment from the lysosomes and its degradation by the proteasome, and with the nuclear translocation of transcription factor EB (TFEB), a transcription factor of many ATG genes. The extra supplementation of proline rescued all these effects.ConclusionsWe demonstrate that the AAR and autophagy are mechanistically linked at the level of mTORC1, and that the lysosome is the central hub of the cross-talk between these two metabolic stress responses.

Regulation and role of the ER stress transcription factor CHOP in alveolar epithelial type-II cells

Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by type-II alveolar epithelial cell (AECII) injury and fibroblast hyperproliferation. Severe AECII endoplasmic reticulum (ER) stress is thought to underlie IPF, but is yet incompletely understood. We studied the regulation of C/EBP homologous protein (CHOP), a proapoptotic ER-stress-related transcription factor (TF) in AECII-like cells. Interestingly, single or combined overexpression of the active ER stress transducers activating transcription factor-4 (Atf4) and activating transcription factor-6 (p50Atf6) or spliced x-box-binding protein-1 (sXbp1) in MLE12 cells did not result in a substantial Chop induction, as compared to the ER stress inducer thapsigargin. Employing reporter gene assays of distinct CHOP promoter fragments, we could identify that, next to the conventional amino acid (AARE) and ER stress response elements (ERSE) within the CHOP promoter, activator protein-1 (AP-1) and c-Ets-1 TF binding sites are necessary for CHOP induction. Serial deletion and mutation analyses revealed that both AP-1 and c-Ets-1 motifs act in concert to induce CHOP expression. In agreement, CHOP promoter activity was greatly enhanced upon combined versus single overexpression of AP-1 and c-Ets-1. Moreover, combined overexpression of AP-1 and c-Ets-1 in MLE12 cells alone in the absence of any other ER stress inducer was sufficient to induce Chop protein expression. Further, AP-1 and c-Ets-1 were upregulated in AECII under ER stress conditions and in human IPF. Finally, Chop overexpression in vitro resulted in AECII apoptosis, lung fibroblast proliferation, and collagen-I production. We propose that CHOP activation by AP-1 and c-Ets-1 plays a key role in AECII maladaptive ER stress responses and consecutive fibrosis, offering new therapeutic prospects in IPF.Key messagesOverexpression of active ER stress sensors Atf4, Atf6, and Xbp1 does not induce Chop.AP-1 and c-Ets-1 TFs are necessary for induction of the ER stress factor Chop.AP-1 and c-Ets-1 alone induce Chop expression in the absence of any ER stress inducers.AP-1 and c-Ets-1 are induced in AECII under ER stress conditions and in human IPF.Chop expression alone triggers AECII apoptosis and consecutive profibrotic responses.

Small regulatory polypeptide of amino acid response negatively relates to poor prognosis and controls hepatocellular carcinoma progression via regulating microRNA-5581-3p/human cardiolipin synthase 1.

Hepatocellular carcinoma (HCC) is one of the most common malignancies with extremely high rates of occurrence and death. Long noncoding RNAs (lncRNAs) have been increasingly revealed to participate in tumorigenesis and development of multiple human cancers, including HCC. LINC00961 is a novel lncRNA which has been uncovered as a tumor suppressor in lung cancer and glioma. However, the role of LINC00961 in HCC has never been probed yet. Herein, we revealed a marked downregulation of LINC00961 in HCC tissues and cell lines. Correlation of low LINC00961 expression with poor outcomes in patients withHCC suggested LINC00961 as an independent predictor for HCC prognosis. Functionally, LINC00961 overexpression obviously inhibited cell proliferation, migration, and invasion in HCC cells. Mechanistically, LINC00961 regulated cardiolipin synthase 1 (CRLS1) expression via sponging miR-5581-3p. Importantly, both miR-5581-3p upregulation and CRLS1 inhibition led to an acceleration in cellular processes in HCC cells. At length, the rescue assays suggested that LINC00961 functioned in HCC through the miR-5581-3p/CRLS1 axis. On the whole, our findings disclosed that LINC00961 played a suppressive role in HCC progression via modulating miR-5581-3p/CRLS1, thus providing a potentially effective target for the prognosis and treatment of patients with HCC.

Plasma metabolomics reveals lower carnitine concentrations in overweight Labrador Retriever dogs

BackgroundThe prevalence of overweight is increasing in dogs, but the metabolic events related to this condition are still poorly understood. The purpose of the study was to investigate the postprandial response of plasma metabolites using a meal-challenge test and to identify metabolic variations related to spontaneous overweightness in privately owned dogs.ResultsTwenty-eight healthy male intact Labrador Retriever dogs were included, 12 of which were classified as lean (body condition score (BCS) 4–5 on a 9-point scale) and 16 as overweight (BCS 6–8). After an overnight fast (14–17 h), blood samples were collected and dogs were thereafter fed a high-fat meal. Postprandial blood samples were collected hourly four times. Plasma metabolites were identified by nuclear magnetic resonance. Postprandial metabolomes differed from the fasting metabolome in multivariate discriminant analysis (PLS-DA: Q2Y = 0.31–0.63, cross-validated ANOVA: P ≤ 0.00014) Eleven metabolites, all amino acids, contributed to the separations. Carnitine was identified as a metabolite related to overweight (stepwise logistic regression analysis P ≤ 0.03) and overweight dogs had overall lower carnitine response (mixed model repeated measures analysis P = 0.005) than lean dogs. Notably, mean fasting carnitine concentration in overweight dogs (9.4 ± 4.2 µM) was close to a proposed reference limit for carnitine insufficiency.ConclusionsA postprandial amino acid response was detected but no time-dependent variations with regards to body condition groups were found. Lower carnitine concentrations were found in overweight compared to lean dogs. The latter finding could indicate a carnitine insufficiency related to spontaneous adiposity and altered lipid metabolism in overweight dogs in this cohort of otherwise healthy Labrador Retrievers.

The effects of maltodextrin and protein supplementation on serum metabolites in exercising competitive weight-pulling dogs

Post-exercise carbohydrate repletion of skeletal muscle glycogen utilising maltodextrin, with or without highly digestible protein, can improve performance in humans which has been extrapolated to dogs. There is limited metabolic evidence regarding substrate utilisation during exercise with and without supplementation other than serum hormone status and serum amino acid responses. The objectives of this study were 2-fold; (1) to examine the metabolomic changes associated with a weight-pulling exercise; and (2) to examine the effects of maltodextrin/protein supplementation on serum metabolomics during recovery. Serum was collected from 12 dogs (6 control and 6 treatment) at different time points (pre-exercise, 0 min post-exercise, 30 min post, 3 h post) and liquid chromatography-mass spectrometry (LC-MS) was performed identifying 242 metabolites. A two-way analysis of variance for time and treatment with false discovery rate correction was performed using MetaboAnalyst 3.0. There were 9 metabolites found to be significantly increased or decreased immediately after exercise from baseline representing primarily citric acid cycle metabolites. Treatment differences at 30 min post-exercise showed increases in 8 metabolites including amino acids and carbohydrate intermediates with supplementation. Thirty-seven metabolites were significantly different at 3 h post-exercise, with most metabolites being related to amino acid increases, as well as decreases in fatty acid metabolites with supplementation. Definite alterations in metabolites suggesting that post-exercise supplementation with maltodextrin and protein supports glucose metabolism and alters fatty acid metabolism or clearance during recovery from a weight-pulling exercise.

Response of Mycorrhiza-Inoculated Pepper and Amino Acids to Salt Treatment at Different Ratios

ABSTRACTMycorrhiza has attracted interest as one of the microorganisms that increase a crop’s salt stress tolerance. This study was conducted to determine the impacts of mycorrhiza inoculation and applying salt at different ratios on the yield of peppers and amino acid concentrations. The study was conducted in greenhouse conditions on loamy soils with four salt treatments, two mycorrhiza inoculations and a control in a complete randomized block design. The present study indicated that salt treatment alone was significantly correlated with crop stem and root amino acid concentrations, RWC% and leaf sizes, whereas applying mycorrhiza showed a positive relationship to stem height, stem and root wet weight, and root amino acids but led to a decloine in root serine and glutamine, and stem amino acid and glutamine. In conclusion, inoculating with mycorrhiza was observed to make a positive contribution to salt stress tolerance at different levels in almost all the parameters examined.

Postprandial Plasma Amino Acid Responses Between Standard Whey Protein Isolate and Whey Protein Isolate Plus Novel Technology.

Background:Muscle mass is an important determinant of metabolic health and physical function. It has previously been demonstrated that the postprandial rise in circulating essential amino acids (EAA) acts as the main stimulus for muscle protein synthesis (MPS). This study investigated postprandial plasma amino acid (AA) responses of 2 different forms of whey protein isolate (WPI) with iso-caloric and iso-nitrogenous profiles to investigate plasma concentrations of EAA.Methods:In all, 12 healthy men (n = 12) between 19 and 32 years of age were recruited for a randomized, cross-over design, which involved consumption of protein supplements on 2 testing days separated by a 6-day washout period between conditions. On each testing day, subjects consumed either 29.6 g of WPI or WPI + io (whey protein isolate plus Ingredient Optimized Protein®) mixed with 236 mL of water. Plasma EAA and branch chain amino acid (BCAA) concentrations were assessed from whole body donated by subjects at pre-consumption and 30, 60, 90, 120, and 180 minutes post consumption.Results:Plasma levels of total EAA concentration was significantly greater in WPI + io at 30, 60, 90, and 120 minutes post consumption (P < .01, P < .001, P < .01, and P < .01, respectively). Plasma levels of total BCAA concentration was significantly greater in WPI + io at 30, 60, 90, and 120 minutes post consumption (P < .01, P < .001, P < .01, and P < .05, respectively) compared with WPI. For leucine, only WPI + io had elevated levels compared with pre-test at 90 minutes post consumption (P < .001).Discussion:Both conditions significantly elevated EAA, BCAA, and leucine from basal levels. However, we conclude that the consumption of the treated WPI significantly raises plasma EAA, BCAA, and leucine to a greater extent compared with WPI with no treatment. Thus, supplementation with WPI that has undergone Ingredient Optimized® technology may be highly beneficial for those who partake in regular exercise, elderly individuals, or those affected by a reduced sensitivity to amino acids.

Postprandial amino acid, glucose and insulin responses among healthy adults after a single intake of Lemna minor in comparison with green peas: a randomised trial.

A high protein content combined with its enormous growth capacity make duckweed an interesting alternative protein source, but information about postprandial responses in humans is lacking. The present study aimed to assess the postprandial serum amino acid profile of Lemna minor in healthy adults in comparison with green peas. A secondary objective was to obtain insights regarding human safety. A total of twelve healthy volunteers participated in a randomised, cross-over trial. Subjects received two protein sources in randomised order with a 1-week washout period. After an overnight fast, subjects consumed L. minor or peas (equivalent to 20 g of protein). After a baseline sample, blood samples were taken 15, 30, 45, 60, 75, 90, 120, 150 and 180 min after consumption to assess amino acid, glucose and insulin levels. Heart rate, blood pressure and aural temperature were measured before and after consumption, and subjects reported on gastrointestinal discomfort for four subsequent days. Compared with green peas, significantly lower blood concentrations of amino acids from L. minor were observed, indicating lower digestibility. L. minor consumption resulted in lower plasma glucose and insulin levels compared with peas, probably due to different glucose content. There were no significant differences concerning the assessed health parameters or the number of gastrointestinal complaints, indicating that a single bolus of L. minor - grown under controlled conditions - did not induce acute adverse effects in humans. Further studies need to investigate effects of repeated L. minor intake and whether proteins purified from L. minor can be digested more easily.

Angiogenesis Induced By Aminoacyl-tRNA Synthetase Deficiency Is Dependent on Amino Acid Response (AAR) but Not Unfolded Protein Response (UPR) Pathways

Stress-induced angiogenesis enormously contributes to both normal development and pathogenesis of various diseases including cancer. Among many stress response pathways implicated in regulation of angiogenesis, the amino acid response (AAR) and the unfolded protein response (UPR) pathways are closely interconnected, as they converge on the common target, eIF2α, which is a key regulator of protein translation. Two kinases, namely Gcn2 (Eif2ak4) and Perk (Eif2ak3), are responsible for transducing signals from AAR and UPR, respectively, to phosphorylation of eIF2α. Even though numerous studies have been performed, this close interconnection between AAR and UPR makes it difficult to clearly distinguish different contributions of these two pathways in regulation of angiogenesis.In this study, we generated a zebrafish angiogenic model harboring a loss-of-function mutation of the threonyl-tRNA synthetase (tars) gene. Tars belongs to a family of evolutionarily conserved enzymes, aminoacyl-tRNA synthetases (aaRSs), which control the first step of protein translation through coupling specific amino acids with their cognate tRNAs. Deficiencies of several aaRSs in zebrafish have been shown to cause increased branching of blood vessels, and this angiogenic phenotype has roughly been explained by activation of AAR and UPR; however, it is unclear whether both AAR and UPR are required and to what extent they contribute to this process. To address this issue, we first performed RNA-seq analyses of Tars-mutated and control zebrafish embryos, as well as those with knockdown of either Gcn2 or Perk in both genotypes. We found that the AAR target genes are dramatically activated in the Tars-mutants, whereas the genes associated with the three UPR sub-pathways (i.e., Perk-, Ire1- and Atf6-mediated pathways) remain inactive, except for very few genes (e.g., Atf3, Atf4, Asns and Igfbp1) that are shared in both AAR and UPR, thus suggesting activation of AAR, but not UPR, in the Tars-mutants. In support of this notion, knockdown of the AAR-associated kinase Gcn2 in the Tars-mutants largely represses the activated genes, while the Perk knockdown shows very little effect. Nonetheless, in contrast to the apparently dispensable role of Perk in Tars-mutants, knockdown of Perk in control embryos leads to specific gene expression alterations, suggesting that Perk effectively functions in homeostatic states (i.e., controls), but, in the stress condition (i.e., Tars-mutants), its function is largely overwhelmed by activation of the Gcn2-mediated AAR.To validate these observations, we investigated the angiogenic phenotypes of the zebrafish models upon genetic and pharmacological interference with the AAR and UPR pathways. A transgenic zebrafish line, Tg(flk1:EGFP), was crossed with the Tars-mutants to visualize angiogenesis in vivo. We observed increased branching of blood vessels in the Tars-mutants, which is rescued by tars mRNA but not an enzymatically dead version. Importantly, knockdown of Gcn2 in the Tars-mutants rescues this phenotype. In contrast, knockdown of Perk, or knockdown of two other known eIF2α kinases, Hri (Eif2ak1) or Pkr (Eif2ak2), shows no effect. Furthermore, knockdown of either one of two major factors downstream to eIF2α, namely Atf4 and Vegfα, or inhibition of Vegf receptor with the drug SU5416, also rescue the phenotype. Thus, these results confirm that AAR, but not UPR, is required for the Tars-deficiency-induced angiogenesis.Taken together, this study demonstrates that, despite being closely interconnected and even sharing a common downstream target, the Gcn2-mediated AAR and the Perk-mediated UPR can be activated independently in different conditions and differentially regulate cellular functions such as angiogenesis. This notion reflects the specificity and efficiency of multiple stress response pathways that are evolved integrally to benefit the organism by ensuring sensing and responding precisely to different types of stresses. This study also provides an example of combining systematic gene expression profiling and phenotypic validations to distinguish activities of such interconnected pathways. Further clarification of the mechanisms shall advance our understanding of how the organisms respond to diverse stresses and how the abnormalities in these regulatory machineries cause cellular stress-related diseases such as cancer, diabetes and immune disorders. DisclosuresNo relevant conflicts of interest to declare.

Digestive Responses to Fortified Cow or Goat Dairy Drinks: A Randomised Controlled Trial.

Fortified milk drinks are predominantly manufactured from bovine (cow) sources. Alternative formulations include those prepared with hydrolysed bovine milk proteins or from alternate bovidae species, such as caprine (goat) milk. Currently, there is little data on protein digestive and metabolic responses following ingestion of fortified milk drinks. To examine the digestive and metabolic responses to commercially-available fortified milks, young adults (n = 15 males: 15 females), in a randomised sequence, ingested isonitrogenous quantities of whole cow-protein (WC), whole goat-protein (WG), or partially-hydrolysed whey cow-protein (HC), commercial fortified milks. Plasma amino acid (AA) and hormonal responses were measured at baseline and again at 5 h after ingestion. Paracetamol recovery, breath hydrogen, and subjective digestive responses were also measured. Postprandial plasma AA was similar between WC and WG, while AA appearance was suppressed with HC. Following HC, there was a negative incremental AUC in plasma branched-chain AAs. Further, HC had delayed gastric emptying, increased transit time, and led to exaggerated insulin and GLP-1 responses, in comparison to whole protein formulas. Overall, WC and WG had similar protein and digestive responses with no differences in digestive comfort. Contrastingly, HC led to delayed gastric emptying, attenuated AA appearance, and a heightened circulating insulin response.

Insights into the Noncoding RNA-encoded Peptides.

Considerable evidence has indicated that most noncoding RNA (ncRNA) transcripts act directly as functional RNAs rather than as encoded peptides. However, a recent study of ribosome occupancy reported that many large intergenic ncRNAs (lincRNAs) are bound by ribosomes, raising the possibility that they are translated into proteins. These lincRNAs contain either Internal Ribosomal Entry Sites (IRES) or short Open Reading Frames (sORFs), and other translation elements, which can be translated into peptides with physiological functions. This review discusses three major types of ncRNA-encoded peptides (ncPEPs). First, microRNA(miRNA)-encoded peptides (miPEPs) are translated from their primary transcripts of miRNA genes and can promote the development of plant roots. Second, a long ncRNA(lncRNA) containing sORF encodes a 90 residues long regulatory peptide. Upon the amino acid response, the lncRNA-encoded peptide reduces the activity of mammalian Target Rapamycin complex 1 (mTORC1) and promotes muscle regeneration. Third, a circular RNA (circRNA) of the Na+/Ca2+ exchanger gene 1 (NCX1) exon 2 transcript encodes a truncated NCX1 protein exhibiting the Na+/Ca2+ exchange activity. It is also of worth noting that the majority of ncRNAs is not translated. This review summarizes the current understanding of the translatability of ncRNAs and the functions of ncPEPs in cellular processes, which may provide novel insights into the roles of ncPEPs.

Timing of post-resistance exercise nutrient ingestion: effects on gastric emptying and glucose and amino acid responses in humans.

This study examined the effects of post-resistance exercise protein ingestion timing on the rate of gastric emptying (GE) and blood glucose (BG) and plasma branched-chain amino acid (BCAA) responses. In all, eleven healthy participants randomly ingested 400 ml of a nutrient-rich drink containing 12 g carbohydrates and 20 g protein at rest (Con), at 5 min (post-exercise (PE)-5) or at 30 min (PE-30) after a single bout of strenuous resistance exercises. The first and second sets comprised ten repetitions at 50 % of each participant's one-repetition maximum (1RM). The third, fourth and fifth sets comprised ten repetitions at 75 % of 1RM, and the sixth set involved repeated repetitions until exhaustion. Following ingestion of the nutrient-rich drink, we assessed the GE rate using 13C-sodium acetate breath test and evaluated two parameters according to the T max-calc (time when the recovery per hour is maximised), which is a standard analytical method, and T 1/2 (time when the total cumulative dose of [13CO2] reaches one-half). T max-calc and T 1/2 were slower for the PE-5 condition than for either the PE-30 or Con condition (T max-calc; Con: 53 (sd 7) min, PE-5: 83 (sd 16) min, PE-30: 62 (sd 9) min, T 1/2; Con: 91 (sd 7) min, PE-5: 113 (sd 21) min, PE-30: 91 (sd 11) min, P<0·05). BG and BCAA responses were also slower for the PE-5 condition than for either the PE-30 or Con condition. Ingesting nutrients immediately after strenuous resistance exercise acutely delayed GE, which affected BG and plasma BCAA levels in blood circulation.

IUGR Is Associated With Marked Hyperphosphorylation of Decidual and Maternal Plasma IGFBP-1.

The mechanisms underpinning intrauterine growth restriction (IUGR), as a result of placental insufficiency, remain poorly understood, no specific treatment is available, and clinically useful biomarkers for early detection are lacking. We hypothesized that human IUGR is associated with inhibition of mechanistic target of rapamycin (mTOR) and activation of amino acid response (AAR) signaling, increased protein kinase casein kinase-2 (CK2) activity, and increased insulin-like growth factor-binding protein 1 (IGFBP-1) expression and phosphorylation in decidua and that maternal plasma IGFBP-1 hyperphosphorylation in the first trimester predicts later development of IUGR. Decidua [n = 16 appropriate-for-gestational age (AGA); n = 16 IUGR] and maternal plasma (n = 13 AGA; n = 13 IUGR) were collected at delivery from two different cohorts. In addition, maternal plasma was obtained in the late first trimester from a third cohort of women (n = 7) who later delivered an AGA or IUGR infant. Total IGFBP-1 expression and phosphorylation (Ser101/Ser119/Ser169), mTOR, AAR, and CK2 activity in decidua and IGFBP-1 concentration and phosphorylation in maternal plasma. We show that decidual IGFBP-1 expression and phosphorylation are increased, mTOR is markedly inhibited, and AAR and CK2 are activated in IUGR. Moreover, IGFBP-1 hyperphosphorylation in first-trimester maternal plasma is associated with the development of IUGR. These data are consistent with the possibility that the decidua functions as a nutrient sensor linking limited oxygen and nutrient availability to increased IGFBP-1 phosphorylation, possibly mediated by mTOR and AAR signaling. IGFBP-1 hyperphosphorylation in first-trimester maternal plasma may serve as a predictive IUGR biomarker, allowing early intervention.

Inhibition of GCN2 sensitizes ASNS-low cancer cells to asparaginase by disrupting the amino acid response

General control nonderepressible 2 (GCN2) plays a major role in the cellular response to amino acid limitation. Although maintenance of amino acid homeostasis is critical for tumor growth, the contribution of GCN2 to cancer cell survival and proliferation is poorly understood. In this study, we generated GCN2 inhibitors and demonstrated that inhibition of GCN2 sensitizes cancer cells with low basal-level expression of asparagine synthetase (ASNS) to the antileukemic agent l-asparaginase (ASNase) in vitro and in vivo. We first tested acute lymphoblastic leukemia (ALL) cells and showed that treatment with GCN2 inhibitors rendered ALL cells sensitive to ASNase by preventing the induction of ASNS, resulting in reduced levels of de novo protein synthesis. Comprehensive gene-expression profiling revealed that combined treatment with ASNase and GCN2 inhibitors induced the stress-activated MAPK pathway, thereby triggering apoptosis. By using cell-panel analyses, we also showed that acute myelogenous leukemia and pancreatic cancer cells were highly sensitive to the combined treatment. Notably, basal ASNS expression at protein levels was significantly correlated with sensitivity to combined treatment. These results provide mechanistic insights into the role of GCN2 in the amino acid response and a rationale for further investigation of GCN2 inhibitors for the treatment of cancer.

Abstract 3847: Inhibition of GCN2 sensitizes ASNS-downregulated cancer cells to asparaginase by disrupting amino acid response

Abstract General control nonderepressible 2 (GCN2) plays a major role in cellular response to amino acid limitation. Although maintenance of amino acid homeostasis is critical for tumor growth, little is known concerning the contribution of GCN2 to cancer cell proliferation and its potential as a therapeutic target. In this study, we demonstrate that inhibition of GCN2 with small-molecule compounds sensitizes cancer cells to anti-leukemic agent asparaginase (ASNase). We first tested acute lymphoblastic leukemia (ALL) cells and showed that treatment with GCN2 inhibitors rendered ALL cells sensitive to ASNase by preventing the induction of asparagine synthetase (ASNS). The prevention of ASNS expression suppresses asparagine synthesis and thus inhibits protein translation. In addition, comprehensive gene expression profiling revealed that the combined treatment induced stress-activated MAPK pathway and thereby triggering apoptosis. Using cell-panel analyses, we also identified a clear synergy between GCN2 inhibitors and ASNase in acute myelogenous leukaemia (AML) and pancreatic cancer cells besides ALL. Notably, basal ASNS expression levels were significantly correlated with sensitivity to combinatorial treatment. Furthermore, the combination displayed robust antitumor activity in mouse xenograft models of ALL, AML, and pancreatic cancer. These results demonstrate a novel mechanistic insight into a role of GCN2 in amino acid response and provide a rationale for further investigating GCN2 inhibitors for the treatment of cancer. Citation Format: Akito Nakamura, Tadahiro Nambu, Shunsuke Ebara, Yuka Hasegawa, Kosei Toyoshima, Yasuko Tsuchiya, Daisuke Tomita, Jun Fujimoto, Osamu Kurasawa, Chisato Takahara, Ayumi Kawamura, Ryuichi Nishigaki, Yoshinori Satomi, Akito Hata, Takahito Hara. Inhibition of GCN2 sensitizes ASNS-downregulated cancer cells to asparaginase by disrupting amino acid response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3847.

Benzylserine inhibits breast cancer cell growth by disrupting intracellular amino acid homeostasis and triggering amino acid response pathways

BackgroundCancer cells require increased levels of nutrients such as amino acids to sustain their rapid growth. In particular, leucine and glutamine have been shown to be important for growth and proliferation of some breast cancers, and therefore targeting the primary cell-surface transporters that mediate their uptake, L-type amino acid transporter 1 (LAT1) and alanine, serine, cysteine-preferring transporter 2 (ASCT2), is a potential therapeutic strategy.MethodsThe ASCT2 inhibitor, benzylserine (BenSer), is also able to block LAT1 activity, thus inhibiting both leucine and glutamine uptake. We therefore aimed to investigate the effects of BenSer in breast cancer cell lines to determine whether combined LAT1 and ASCT2 inhibition could inhibit cell growth and proliferation.ResultsBenSer treatment significantly inhibited both leucine and glutamine uptake in MCF-7, HCC1806 and MDA-MB-231 breast cancer cells, causing decreased cell viability and cell cycle progression. These effects were not primarily leucine-mediated, as BenSer was more cytostatic than the LAT family inhibitor, BCH. Oocyte uptake assays with ectopically expressed amino acid transporters identified four additional targets of BenSer, and gas chromatography-mass spectrometry (GCMS) analysis of intracellular amino acid concentrations revealed that this BenSer-mediated inhibition of amino acid uptake was sufficient to disrupt multiple pathways of amino acid metabolism, causing reduced lactate production and activation of an amino acid response (AAR) through activating transcription factor 4 (ATF4).ConclusionsTogether these data showed that BenSer blockade inhibited breast cancer cell growth and viability through disruption of intracellular amino acid homeostasis and inhibition of downstream metabolic and growth pathways.

Effects of Cold Shock on Responses of Phosphomonoesters and Free Amino Acids in Phospholipid-Rich Organs in the Amur Sleeper Perccottus Glehni

Similarities and differences were found in the mechanisms of adaptation of the brain and liver to cold shock in the freshwater fish Perccottus glehni. The quantity of phosphoethanolamine in the brain on day 4 of cold shock at +4°C increased 84-fold from the level in controls (+20°C), accounting for 22.3% of the total pool of free amino acids and ninhydrin-positive compounds. Phosphoserine was absent from the brain at +20°C, but cold shock initiated its accumulation to 1.8% of the total pool. The taurine pool at +4°C decreased from 28.3 to 20% of the total pool, yielding first place to phosphoethanolamine. However, in the liver, phosphoethanolamine (PE) and phosphoserine (PS) were not seen in the free form either in controls or at 4°C. The dominant amino acid in the liver at both temperatures was taurine, the quantity at +4°C being significantly increased. Th glutamate pool decreased nine-fold at +4°C. It is suggested that the intense accumulation of PE and PS in the brain at low temperatures is evidence of the specific features of the adaptation of the neuron membrane, reflecting quantitative changes in phospholipids, possibly including sphingosine-1-phosphate.

Variations in free amino acid concentrations in mosses and different parts of Cinnamomum camphora along an urban-to-rural gradient

Vegetation growing in urban ecosystems is frequently exposed to an environment with high atmospheric nitrogen (N) pollution. We systematically investigated the free amino acid concentrations in moss samples and Cinnamomum camphora leaf (new, middle-aged and old leaves), branch phloem, trunk phloem, and bark samples from field sites (Guiyang City, SW China) with different N deposition levels. The responses of the free amino acids to N deposition were analysed in the abovementioned plant tissues to determine whether plant free amino acids could act as biomarkers of the external N supply; moreover, differences in the N metabolism of these tissues under varying N deposition conditions were revealed by the compositions and sizes of their free amino acid pools. In particular, we reported the significant accumulation of arginine with increased N deposition in bark samples (while the arginine concentrations in the branch phloem, trunk phloem, and leaves remained low), which may indicate a long-term or historic external environment with a high N availability; additionally, the noticeable dominance and fluctuation of γ-aminobutyric acid in response to varied N deposition levels occurred in both the branch phloem and the trunk phloem, suggesting that the γ-aminobutyric acid transported in the phloem may be used as an important signal reflecting increases in the atmospheric N input. We conclude that the free amino acid concentrations in moss and camphor leaf, phloem and bark tissues are more sensitive to N deposition compared to their N concentrations and that tissue glutamine/glutamate and arginine/γ-aminobutyric acid ratios may serve as better biomarkers reflecting the tissue N accumulation status associated with increased N deposition. Therefore, free amino acid concentration analyses of different plant parts may provide a means to gain a more in-depth understanding of the impacts of atmospheric N pollution on plant physiology and N cycles.

Doxorubicin chemotherapy affects the intracellular and interstitial free amino acid pools in skeletal muscle.

Skeletal muscle (SM) health and integrity is dependent on the dynamic balance between protein synthesis and degradation, and central to this process is the availability of amino acids (AA) in the amino pool. While Doxorubicin (DOX) remains one of the most widely used chemotherapeutic agents for the treatment of solid and hematological malignancies, little is known of the effect of the drug on SM, particularly its effect on the availability of amino acids in the tissue. The purpose of this study was to examine the effect of DOX administration on vascular, interstitial and intracellular concentrations of AA in SM of the rat up to 8 days after the administration of a 1.5 or 4.5 mg/kg i.p. dose of DOX. In the plasma, total amino acids (TAA) were significantly increased compared to control where greater (P<0.05) concentrations were observed following the 1.5 mg/kg dose compared to the 4.5 mg/kg dose. Compared to control, the 1.5 mg/kg dose resulted in an increase (P<0.05) in interstitial TAA whereas the 4.5 mg/kg resulted in a sustained decrease (P<0.05). Intracellular TAA, essential amino acids (EAA) and branched-chain amino acids (BCAA) where significantly increased in each muscle group analyzed, following the 1.5 and 4.5 mg/kg doses compared to control. This study provides important insight into the amino acid response following DOX chemotherapy and presents a substantial foundation for future studies focused on reducing SM damage and recovery by targeting amino acid metabolism.