Expression Of Amino Acid Transporters Research Articles

Zinc proteinate with moderate chelation strength enhances zinc absorption by up-regulating the expression of zinc and amino acid transporters in primary cultured duodenal epithelial cells of broiler embryos.

Recent study showed that zinc (Zn) and amino acid transporters may be involved in enhancing Zn absorption from Zn proteinate with moderate chelation strength (Zn-Prot M) in the duodenum of broilers. However, the specific mechanisms by which Zn-Prot M promotes the above Zn absorption are unknown. Therefore, in this study, three experiments were conducted to investigate specific and direct effects of Zn-Prot M and Zn sulfate (ZnS) on Zn absorption and expression of related transporters in primary duodenal epithelial cells of broiler embryos so as to preliminarily address possible mechanisms. In experiment 1, cells were treated with 100 μmol Zn/L as ZnS or Zn-Prot M for 20, 40, 60, 80, 100 or 120min. Experiment 2 consisted of 3 sub-experiments. In experiment 2A, cells were treated with a Zn-unsupplemented basal medium (Control) or the basal medium supplemented with 100 or 200 μmol Zn/L as ZnS or Zn-Prot M for 60min; in experiment 2B, cells were treated with a Zn-unsupplemented basal medium (Control) or the basal medium supplemented with 200 μmol Zn/L of as the ZnS or Zn-Prot M for 120min; in experiment 2C, cells were treated with a Zn-unsupplemented basal medium (Control) or the basal medium supplemented with 400 or 800 μmol Zn/L as ZnS or Zn-Prot M for 120min. In experiment 3, cells were treated with a Zn-unsupplemented basal medium (Control) or the basal medium supplemented with 400 μmol Zn/L as ZnS or Zn-Prot M for 120min. The results of experiment 1 indicated that the minimum incubation time for saturable Zn absorption was determined to be 50.83min using the best fit line. The results in experiment 2 demonstrated that a Zn concentration of 400 μmol/L and an incubation time of 120min were suitable to increase the absorption of Zn from Zn-Prot M compared to ZnS. In experiment 3, Zn absorption across cell monolayers was significantly increased by Zn addition (P < 0.05), and was significantly greater with Zn-Prot M than with ZnS (P < 0.05). Compared to the control, Zn addition significantly decreased Zn transporter 10 and peptide-transporter 1 mRNA expression levels and increased y+L-type amino transporter 2 (y+LAT2) protein abundance (P < 0.05). Moreover, protein expression levels of zrt/irt-like protein 3 (ZIP3), ZIP5 and y+LAT2 were significantly greater for Zn-Prot M than for ZnS (P < 0.05). These findings suggest that Zn-Prot M promote Zn absorption by increasing ZIP3, ZIP5 and y+LAT2 protein expression levels in primary duodenal epithelial cells.

Reprogramming of Glutamine Amino Acid Transporters Expression and Prognostic Significance in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the most prevalent primary liver malignancy and is a major cause of cancer-related mortality in the world. This study aimed to characterize glutamine amino acid transporter expression profiles in HCC compared to those of normal liver cells. In vitro and in vivo models of HCC were studied using qPCR, whereas the prognostic significance of glutamine transporter expression levels within patient tumors was analyzed through RNAseq. Solute carrier (SLC) 1A5 and SLC38A2 were targeted through siRNA or gamma-p-nitroanilide (GPNA). HCC cells depended on exogenous glutamine for optimal survival and growth. Murine HCC cells showed superior glutamine uptake rate than normal hepatocytes (p &lt; 0.0001). HCC manifested a global reprogramming of glutamine transporters compared to normal liver: SLC38A3 levels decreased, whereas SLC38A1, SLC7A6, and SLC1A5 levels increased. Also, decreased SLC6A14 and SLC38A3 levels or increased SLC38A1, SLC7A6, and SLC1A5 levels predicted worse survival outcomes (all p &lt; 0.05). Knockdown of SLC1A5 and/or SLC38A2 expression in human Huh7 and Hep3B HCC cells, as well as GPNA-mediated inhibition, significantly decreased the uptake of glutamine; combined SLC1A5 and SLC38A2 targeting had the most considerable impact (all p &lt; 0.05). This study revealed glutamine transporter reprogramming as a novel hallmark of HCC and that such expression profiles are clinically significant.

Methionine Source and Level Modulate Gut pH, Amino Acid Transporters and Metabolism Related Genes in Broiler Chickens.

This study determined the effects of two methionine (Met) sources at three total sulfur amino acids (TSAA) to lysine ratios (TSAA/Lys) on gut pH, digestive enzyme activity, amino acid transporter expression, and Met metabolism of broilers. The birds were randomly assigned to a 2 × 3 factorial arrangement with Met sources (dl-Met and dl-2-hydroxy-4-(methylthio)-butanoic acid (OH-Met)) and TSAA/Lys (0.58, 0.73, and 0.88) from 1 to 21 days. The results demonstrated that dl-Met and OH-Met supported the same growth performance, but high TSAA/Lys ratio reduced the feed intake and body weight (P < 0.05). OH-Met reduced the crop chyme pH and enhanced the jejunal lipase activity (P < 0.05). ATB0,+ expression decreased with increased dl-Met levels in the duodenum; the low TSAA/Lys ratio induced a stronger mRNA expression of basolateral Met transporters. OH-Met resulted in an increase of cystathionine β-synthase expression in the liver and a decrease in serum homocysteine levels at middle TSAA/Lys ratio compared with dl-Met treatment (P < 0.05). In conclusion, two Met sources support the same growth, but OH-Met acidified the crop chyme. The investigated transporter transcripts differed significantly along the small intestine. At the middle TSAA/Lys ratio, OH-Met showed a higher metabolic tendency of the trans-sulfuration pathway compared with dl-Met.

Dexamethasone alleviates etomidate-induced myoclonus by reversing the inhibition of excitatory amino acid transporters.

Etomidate can induce myoclonus with an incidence of 50 ~ 85% during anesthesia induction. Dexamethasone, as a long-acting synthetic glucocorticoid, has neuroprotective effects. However, the effects of dexamethasone on the etomidate-induced myoclonus remain uncertain. Adult male Sprague-Dawley rats were randomly assigned to receive etomidate (1.5 mg/kg) plus dexamethasone (4 mg/kg) (etomidate plus dexamethasone group) or etomidate (1.5 mg/kg) plus the same volume of normal saline (NS) (etomidate plus NS group). The mean behavioral scores, local field potentials and muscular tension were recorded to explore the effects of dexamethasone on etomidate-induced myoclonus. Liquid chromatography coupled with tandem mass spectrometric system (LC-MS/MS), quantitative real-time polymerase chain reaction (qRT-PCR), and western blotting were applied to analyze the levels of glutamate and γ-aminobutyric acid (GABA), the mRNA and protein expression of excitatory amino acid transporters (EAATs), and plasma corticosterone levels at different time points after anesthesia. Compared with the etomidate plus NS treatment, the etomidate plus dexamethasone treatment significantly decreased the mean behavioral score at 1, 3, 4, and 5 min after administration; the peak power spectral density (PSD) (p = 0.0197) in the analysis of ripple waves; and the glutamate level (p = 0.0139) in the neocortex. However, compared with etomidate plus NS, etomidate plus dexamethasone increased the expression of the neocortical proteins of EAAT1 (p = 0.0207) and EAAT2 (p = 0.0022) and aggravated the inhibition of corticosterone at 4 h (p = 0.0019), 5 h (p = 0.0041), and 6 h (p = 0.0009) after administration. Dexamethasone can attenuate the myoclonus, inhibit the glutamate accumulation, and reverse the suppression of EAATs in the neocortex induced by etomidate following myoclonus, while conversely aggravating etomidate-induced adrenal suppression.

ATF4 as a Prognostic Marker and Modulator of Glutamine Metabolism in Oestrogen Receptor-Positive Breast Cancer.

ATF4, a stress-responsive transcription factor that upregulates adaptive genes, is a potential prognostic marker and modulator of glutamine metabolism in breast cancer. However, its exact role remains to be elucidated. ATF4 expression was evaluated at genomic and transcriptomic levels using METABRIC (n = 1,980), GeneMiner (n = 4,712), and KM-Plotter datasets. Proteomic expression was assessed via immunohistochemistry (n = 2,225) in the Nottingham Primary Breast Cancer Series. ATF4 genomic copy number (CN) variation and mRNA/protein in association with clinicopathological parameters, amino acid transporters (AATs), and patient outcome were investigated. Genomic, transcriptomic, and proteomic overexpression of ATF4 was associated with more aggressive ER-negative tumours. ATF4 mRNA and protein expression were significantly associated with increased expression of glutamine related AATs including SLC1A5 (p &lt; 0.01) and SLC7A11 (p &lt; 0.02). High ATF4 and SLC1A5 protein expression was significantly associated with shorter breast cancer-specific survival (p &lt; 0.01), especially in ER+ tumours (p &lt; 0.01), while high ATF4 and SLC7A11 protein expression was associated with shorter survival (p &lt; 0.01). These findings suggest a complex interplay between ATF4 and AATs in breast cancer biology and underscore the potential role for ATF4 as a prognostic marker in ER+ breast cancer, offering a unique opportunity for risk stratification and personalized treatment strategies.

Functional Impact of nTS Glutamate Stress on Respiration in an Alzheimer’s Disease Model

Alzheimer’s disease (AD) is closely associated with obstructive sleep apnea (OSA). Such hypoxic insults trigger glutamate release of chemoafferents into the nucleus tractus solitarii (nTS), an important upstream center of the chemoreflex. Potential alterations of glutamate handling in the nTS may lead to the respiratory dysfunction seen in AD patients. Using the streptozotocin (STZ)-induced rat model — an effective proxy for human AD — we studied the functional consequences of nTS glutamate stress on respiration. STZ-AD was induced in 6-week-old male Sprague Dawley rats via intracerebroventricular injections of 2 - 2.25 mg/kg STZ. After two weeks, EMG recordings of diaphragm activity served as surrogate for respiratory responses to glutamate microinjections (20 nL of 40 mM) into the caudal nTS. In a subset of rats, chemoafferent terminals were labeled with the fluorescent tracer DiI to permit patch clamp recordings of identified 2nd order nTS neurons participating in the chemoreflex loop. Evoked glutamatergic excitatory postsynaptic currents (TS-EPSCs) were generated via tractus solitarii stimulation at frequencies (10 - 40 Hz) typical for afferent discharge during hypoxia. Acute glutamate injections into the caudal nTS increased respiratory frequency by 15 - 20 breaths per minute. The response magnitude was similar between CTL and STZ-AD animals. Excitatory stress with repeated glutamate injections (5 min. apart) evoked a reliable respiratory response in CTL that slowly declined to ~65% from its maximum over the course of 10 injections. In contrast, the decline of respiratory response in STZ-AD was more pronounced and occurred significantly earlier than in CTL, indicating altered glutamate handling in the caudal nTS of STZ-AD rats. Next, we used patch clamp electrophysiology to analyze increased glutamate release of chemoafferents onto 2nd order nTS neurons. High frequency stimulation of chemoafferents induced a successive depression of TS-EPSC amplitude in nTS neurons. TS-EPSC depression was significantly stronger in STZ-AD and may be due to altered glutamate handling at the synapse. Western Blot analysis of STZ-AD brainstem tissue including the nTS revealed a significant increase in glial fibrillary acidic protein (GFAP) expression and a decrease of excitatory amino acid transporters (EAATs) expression, suggesting astrocyte involvement in compromised nTS glutamate handling in STZ-AD. In summary, the STZ-AD model showed reduced respiratory responses to glutamate stress in the caudal nTS. The altered stress response may come from enhanced depression of TS-EPSC amplitude at nTS neurons in the chemoreflex. Altered astrocytic glutamate removal from the synaptic cleft may contribute to TS-EPSC depression. Together, altered glutamate handling in a respiratory brainstem center may contribute to OSA in AD patients. NIH R15AG065927 (TDO &amp; DO), KCOM Biomedical Graduate Program (RKT &amp;TDO), ATSU Research Support (SKRC). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Increased excitatory amino acid transporter 2 levels in basolateral amygdala astrocytes mediate chronic stress-induced anxiety-like behavior

Abstract The conventional perception of astrocytes as mere supportive cells within the brain has recently been called into question by empirical evidence, which has revealed their active involvement in regulating brain function and encoding behaviors associated with emotions. Specifically, astrocytes in the basolateral amygdala (BLA) have been found to play a role in the modulation of anxiety-like behaviors triggered by chronic stress. Nevertheless, the precise molecular mechanisms by which BLA astrocytes regulate chronic stress–induced anxiety-like behaviors remain to be fully elucidated. In this study, we found that in a mouse model of anxiety triggered by unpredictable chronic mild stress (UCMS), the expression of excitatory amino acid transporter 2 (EAAT2) was upregulated in the BLA. Interestingly, our findings indicate that the targeted knockdown of EAAT2 specifically within the BLA astrocytes was able to rescue the anxiety-like behavior in mice subjected to stress. Furthermore, we found that the overexpression of EAAT2 in the BLA, whether achieved through intracranial administration of EAAT2 agonists or through injection of EAAT2-overexpressing viruses with GfaABC1D promoters, evoked anxiety-like behavior in mice. Our single-nucleus RNA sequencing analysis further confirmed that chronic stress induced an upregulation of EAAT2 specifically in astrocytes in the BLA. Moreover, through in vivo calcium signal recordings, we found that the frequency of calcium activity in the BLA of mice subjected to chronic stress was higher compared with normal mice. After knocking down the expression of EAAT2 in the BLA, the frequency of calcium activity was not significantly increased, and anxiety-like behavior was obviously mitigated. Additionally, administration of an EAAT2 inhibitor in the BLA yielded a notable reduction in anxiety level among mice subjected to stress. These results suggest that BLA astrocytic EAAT2 plays a role in in the regulation of UCMS-induced anxiety-like behavior by impacting the activity of local glutamatergic neurons, and targeting EAAT2 in the BLA holds therapeutic promise for addressing anxiety disorders.

Effect of L-Type Amino Acid Transporter 1 Expression on Clinicopathological Features and Prognosis of Non-Hodgkin's Lymphoma

To detect the expression of L-type amino acid transporter 1 (LAT1) in non-Hodgkin's lymphoma (NHL) tissues, and analyze its effect on clinicopathological characteristics and prognosis of patients. A total of 92 NHL patients who were treated in our hospital from January 2017 to April 2019 were collected. The expression of LAT1 in NHL tissue was detected by immunohistochemistry and compared between patients with different pathological features (including sex, Ann Arbor stage, extranodal infiltration, Ki-67). The risk factors affecting mortality were analyzed using univariate and multivariate Cox proportional hazards regression. Receiver operating characteristic (ROC) curve was used to detect the predictive value of percentage of LAT1-positive cells in NHL tissue for patient mortality, and analyzing the effect of percentage of LAT1-positive cells on survival rate. LAT1 was positively expressed in NHL tissue. The high expression rate of LAT1 in Ann Arbor stage III and IV groups were higher than that in Ann Arbor stage I group, that in extranodal infiltration group was higher than non-extranodal infiltration group, and that in Ki-67 positive expression group was higher than Ki-67 negative expression group (all P < 0.05). The remission rate after 3 courses of treatment in high-LAT1 expression group was 70.7%, which was lower than 91.2% in low-LAT1 expression group (P < 0.05). Ann Arbor stage III and IV, extranodal invasion, Ki-67 positive expression and increased expression of LAT1 (LAT1-positive cell percentage score ≥2) were risk factors for mortality. The cut-off value of percentage of LAT1-positive cells for predicting NHL death was 45.6%, and the area under the ROC curve was 0.905 (95%CI: 0.897-0.924). The 3-year survival rate of high-LAT1 level group (the percentage of LAT1-positive cells≥45.6%) was 50.00%, which was lower than 78.26% of low-LAT1 level group (P < 0.05). The expression level of LAT1 in NHL tissue increases, which affects Ann Arbor stage and extranodal infiltration of patients. LAT1 is a risk factor for death.

Abstract 7052: p53 underpins a dependence on oxidative phosphorylation in glycolysis-competent colorectal cancer

Abstract The use of mitochondrial inhibitors to target oxidative phosphorylation (OXPHOS) in cancer treatment presents a challenge due to dose-limiting toxicities. Moreover, while glycolysis-deficient cancers are vulnerable to OXPHOS inhibition in preclinical models, the full extent of phenotypical and mechanistic consequences of inhibiting OXPHOS in cancers capable of glycolysis is not yet well understood. Our results presented here offer promising insights into potential therapeutic gains from combining p53 restoration strategies with OXPHOS inhibitors, even when applied to glycolysis-competent CRC cells. Treatment with mitochondrial complex I inhibitors did not cause energy stress in CRC cells capable of glycolysis. It does, however, induce DNA replication stress, apparent through an observed cell cycle arrest at the S phase, enrichment of the G2/M DNA-damage checkpoint regulation pathway, and replication fork slowdown. Intriguingly, CRC cells harboring wildtype p53 exhibited more severe replication stress than those carrying mutant p53. Furthermore, siRNA knockdown of p53 attenuates replication stress and reduces cell cycle arrest, underlining the important role of p53 in CRC cell responses to OXPHOS inhibition. Our targeted metabolomics analysis reveals that OXPHOS inhibition results in reductions in the purine nucleotides, adenine monophosphate (AMP) and guanine monophosphate (GMP), as well as the pyrimidine nucleotide, uridine monophosphate (UMP), in CRC cells, regardless of their p53 mutational status. By supplementing cell culture mediums with the purine nucleobases adenine and guanine, and the pyrimidine nucleoside uridine, we observed a partial reversal of the replication fork slowdown and reductions in cell viability. This suggests nucleotide deficiencies are involved in the induction of DNA replication stress caused by OXPHOS inhibition. The nucleotide deficiencies were associated with a decrease in the nucleobase precursor aspartate. By adding aspartate at a supraphysiological concentration, to overcome the low expression of the excitatory amino acid transporter 1 required for cellular import of aspartate, we were able to restore the levels of nucleotides. Collectively, our findings suggest broader potential cancer treatment paradigms via OXPHOS targeting, extending beyond glycolysis-deficient cancers. Our data uncovers that CRC cells, which commonly exhibit the glycolytic phenotype, are susceptible to OXPHOS inhibition, with those carrying wildtype p53 showing heightened sensitivity. Therefore, p53 status could serve as a biomarker for predicting CRC responses to OXPHOS inhibitors. Moreover, our findings suggest that combined strategies of restoring p53 function, using small molecules such as APR-246, might enable reduced dosage of OXPHOS inhibitors in CRC treatment, thereby mitigating their dose-limiting toxicities. Citation Format: Xiao Hong Zhao, Man Man Han, Qian Qian Yan, Yi Meng Yue, Kai Hong Ye, Yuan Yuan Zhang, Liu Teng, Liang Xu, Xiao Jing Shi, Ting La, Yu Chen Feng, Ran Xu, Vinod K. Narayana, David P. De Souza, Tao Liu, Mark Baker, Rick F. Thorne, Xu Dong Zhang, Song Chen, Lei Jin. p53 underpins a dependence on oxidative phosphorylation in glycolysis-competent colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7052.

Abstract 388: Identifying synthetic lethalities in GCN2 KO tumors using an in vivo targeted CRISPRi screen

Abstract The integrated stress response (ISR) is an intracellular signaling pathway that fosters cell adaptation to diverse stresses. The ISR is mediated by a family of four eIF2 kinases that each sense distinct cellular stresses. Activation of the eIF2 kinases results in reprogrammed gene expression through translational and transcriptional mechanisms that help prevent stress damage. Previously, we reported that prostate cancer (PCa) cells rely on GCN2 eIF2 kinase for maintenance of amino acid (AA) homeostasis to sustain growth (Cordova et al., 2022 eLife 11:e81083). GCN2 inhibition through pharmacological or genetic means results in decreased expression of AA transporters, depletion of intracellular AAs, and reduced growth of PCa cells in culture and mouse xenograft models. Although loss of GCN2 results in a significant reduction in PCa cell growth, minimal cell death is observed. We hypothesize that loss of GCN2 in PCa cells results in vulnerabilities that can be exploited and targeted to induce cell death. To optimize GCN2-targeted therapies for PCa, we utilized a targeted CRISPR-interference (CRISPRi) screen in castration-resistant 22Rv1 cells with functional wild type (WT) GCN2 or those with it deleted (GCN2 KO). The CRISPRi screen was performed in cells grown in culture or grown as tumor xenografts in mice and featured a guide library targeting genes that encode druggable targets, including kinases, phosphatases, and metabolic enzymes. While reduced expression of 152 genes was synthetically lethal with loss of GCN2 in the in vitro screen, more genes (202) were synthetically lethal with loss of GCN2 in vivo. Of note, reduced expression of many metabolic enzymes was synthetically lethal with GCN2 ablation only in the in vivo screen, potentially due to the abundance of nutrients in standard culture media. A group of six metabolism-related genes, identified as hits in the in vivo screen and targets of FDA-approved drugs, were selected for follow-up studies. One of these metabolic enzymes was GART, a trifunctional enzyme that carries out 3 of the 10 enzymatic steps in de novo purine biosynthesis. Based on our findings showing that GCN2 maintains AA levels to support nucleotide pools in PCa cells, we initially focused on this critical enzyme. Inhibition of GART using antifolates, including the FDA-approved Pemetrexed, resulted in minimal cell death of parental and GCN2 KO PCa cells cultured in standard culture media. However, when cultured in reduced nutrient media that better mimicked reduced nutrient availability in vivo, GART inhibition resulted in selective death of GCN2 KO cells. Our CRISPRi screening strategy uncovers new vulnerabilities in GCN2-inhibited PCa cells and reveals a general strategy to identify actionable targets with the potential to combine with GCN2 inhibitors for the treatment of PCa. Citation Format: Noah R. Sommers, Ricardo A. Cordova, Angela J. Klunk, Ronald C. Wek, Kirk A. Staschke. Identifying synthetic lethalities in GCN2 KO tumors using an in vivo targeted CRISPRi screen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 388.

Investigation of the Effects of a Novel NOX2 Inhibitor, GLX7013170, against Glutamate Excitotoxicity and Diabetes Insults in the Retina.

Glutamate excitotoxicity and oxidative stress represent two major pathological mechanisms implicated in retinal disorders. In Diabetic Retinopathy (DR), oxidative stress is correlated to NADPH oxidase (NOX), a major source of Reactive Oxygen Species (ROS), and glutamate metabolism impairments. This study investigated the role of NOX2 and the novel NOX2 inhibitor, GLX7013170, in two models of a) retinal AMPA excitotoxicity [AMPA+GLX7013170 (10-4 M, intravitreally)] and b) early-stage DR paradigm (ESDR), GLX7013170: 14-day therapeutic treatment (topically, 20 μL/eye, 10 mg/mL (300 × 10-4 M), once daily) post-streptozotocin (STZ)-induced DR. Immunohistochemical studies for neuronal markers, nitrotyrosine, micro/macroglia, and real-time PCR, Western blot, and glutamate colorimetric assays were conducted. Diabetes increased NOX2 expression in the retina. NOX2 inhibition limited the loss of NOS-positive amacrine cells and the overactivation of micro/macroglia in both models. In the diabetic retina, GLX7013170 had no effect on retinal ganglion cell axons, but reduced oxidative damage, increased Bcl-2, reduced glutamate levels, and partially restored excitatory amino acid transporter (EAAT1) expression. These results suggest that NOX2 in diabetes is part of the triad, oxidative stress, NOX, and glutamate excitotoxicity, key players in the induction of DR. GLX7013170 is efficacious as a neuroprotective/anti-inflammatory agent and a potential therapeutic in retinal diseases, including ESDR.

Effects of the kinetic pattern of dietary glucose release on nitrogen utilization, the portal amino acid profile, and nutrient transporter expression in intestinal enterocytes in piglets

BackgroundPromoting the synchronization of glucose and amino acid release in the digestive tract of pigs could effectively improve dietary nitrogen utilization. The rational allocation of dietary starch sources and the exploration of appropriate dietary glucose release kinetics may promote the dynamic balance of dietary glucose and amino acid supplies. However, research on the effects of diets with different glucose release kinetic profiles on amino acid absorption and portal amino acid appearance in piglets is limited. This study aimed to investigate the effects of the kinetic pattern of dietary glucose release on nitrogen utilization, the portal amino acid profile, and nutrient transporter expression in intestinal enterocytes in piglets.MethodsSixty-four barrows (15.00 ± 1.12 kg) were randomly allotted to 4 groups and fed diets formulated with starch from corn, corn/barley, corn/sorghum, or corn/cassava combinations (diets were coded A, B, C, or D respectively). Protein retention, the concentrations of portal amino acid and glucose, and the relative expression of amino acid and glucose transporter mRNAs were investigated. In vitro digestion was used to compare the dietary glucose release profiles.ResultsFour piglet diets with different glucose release kinetics were constructed by adjusting starch sources. The in vivo appearance dynamics of portal glucose were consistent with those of in vitro dietary glucose release kinetics. Total nitrogen excretion was reduced in the piglets in group B, while apparent nitrogen digestibility and nitrogen retention increased (P < 0.05). Regardless of the time (2 h or 4 h after morning feeding), the portal total free amino acids content and contents of some individual amino acids (Thr, Glu, Gly, Ala, and Ile) of the piglets in group B were significantly higher than those in groups A, C, and D (P < 0.05). Cluster analysis showed that different glucose release kinetic patterns resulted in different portal amino acid patterns in piglets, which decreased gradually with the extension of feeding time. The portal His/Phe, Pro/Glu, Leu/Val, Lys/Met, Tyr/Ile and Ala/Gly appeared higher similarity among the diet treatments. In the anterior jejunum, the glucose transporter SGLT1 was significantly positively correlated with the amino acid transporters B0AT1, EAAC1, and CAT1.ConclusionsRational allocation of starch resources could regulate dietary glucose release kinetics. In the present study, group B (corn/barley) diet exhibited a better glucose release kinetic pattern than the other groups, which could affect the portal amino acid contents and patterns by regulating the expression of amino acid transporters in the small intestine, thereby promoting nitrogen deposition in the body, and improving the utilization efficiency of dietary nitrogen.

Anesthetic Sevoflurane Induces Enlargement of Dendritic Spine Heads in Mouse Neurons via Tau-Dependent Mechanisms.

Sevoflurane induces neuronal dysfunction and cognitive impairment. However, the underlying mechanism remains largely to be determined. Tau, cyclophilin D, and dendritic spine contribute to cognitive function. But whether changes in dendritic spines are involved in the effects of sevoflurane and the potential association with tau and cyclophilin D is not clear. We harvested hippocampal neurons from wild-type mice, tau knockout mice, and cyclophilin D knockout mice. We treated these neurons with sevoflurane at day in vitro 7 and measured the diameter of dendritic spine head and the number of dendritic spines. Moreover, we determined the effects of sevoflurane on the expression of excitatory amino acid transporter 3 (EAAT3), extracellular glutamate levels, and miniature excitatory postsynaptic currents (mEPSCs). Finally, we used lithium, cyclosporine A, and overexpression of EAAT3 in the interaction studies. Sevoflurane-induced tau phosphgorylation increased the diameter of dendritic spine head and decreased the number of dendritic spines in neurons harvested from wild-type and cyclophilin D knockout mice, but not tau knockout mice. Sevoflurane decreased the expression of EAAT3, increased extracellular glutamate levels, and decreased the frequency of mEPSCs in the neurons. Overexpression of EAAT3 mitigated the effects of sevoflurane on dendritic spines. Lithium, but not cyclosporine A, attenuated the effects of sevoflurane on dendritic spines. Lithium also inhibited the effects of sevoflurane on EAAT3 expression and mEPSCs. These data suggest that sevoflurane induces a tau phosphorylation-dependent demtrimental effect on dendritic spine via decreasing EAAT3 expression and increasing extracellular glutamate levels, leading to neuronal dysfunction.

Shifts in microbiota and gene expression of nutrient transporters, mucin and interleukins in the gut of fast-growing and slow-growing chickens infected by Salmonella Enteritidis.

Slow-growing breeds are more resistant to Salmonella infection compared to fast-growing broilers. However, it is unclear whether that is associated with innate resistance or rather rely on differences in Salmonella-induced gut responses. We investigated the microbial composition and gene expression of nutrient transporters, mucin, and interleukin in the gut of a fast-growing (Cobb500) and a slow-growing naked neck (NN) chicken breeds challenged with Salmonella Enteritidis. Hatchlings were inoculated at two days of age using sterile broth (sham) or Salmonella Enteritidis (SE) and distributed according to a completely randomized design into four treatments: Cobb-sham; Cobb-SE; NN-sham; and NN-SE. Cecal SE counting and microbial composition by 16S rRNA sequencing were determined at 24-, 96-, and 168-hours post-inoculation (hpi). Gene expression of amino acid (Asct1) and peptide transporters (PepT1), glucose transporters (Sglt1, Glut2 and Glut5) and mucin (Muc2) in the jejunum and expression of interleukins (IL1 beta, IL8, IL17 and IL22) in the cecum was assessed by qPCR at 24 and 168 hpi. NN birds were colonized by SE just as Cobb birds but showed innate upregulation of Muc2, IL8 and IL17 in comparison to Cobb. While nutrient transporter mRNA expression was impaired in SE-challenged Cobb birds, the opposite was observed in NN. There were no differences in microbial diversity at different sampling times for Cobb-SE, whereas the other groups had higher diversity and lower dominance at 24 hpi compared with 96 hpi and 168 hpi. NN birds apparently develop earlier gut microbial stability, have higher basal level of mucin gene expression as well as differential nutrient transporter and interleukin gene expression in the presence of SE which might mitigate the effects of SE infection compared to Cobb birds.

Apparent ileal amino acid digestibility, gut morphometrics, and gene expression of peptide and amino acid transporters in broiler chickens fed low-crude-protein diets supplemented with crystalline amino acids with soybean meal, canola meal, or corn DDGS as protein feedstuffs.

We investigated the impact of using canola meal (CM) or corn distillers dried grain soluble (cDDGS) in place of soybean meal (SBM) in low-crude-protein diets supplemented with amino acids (AA) on AA digestibility, gut morphometrics, and AA transporter genes in broiler chicken. On day 0, 540 Cobb 500 male broilers were allocated to six diets in 36-floor pens. The positive control (PC) was a corn-SBM diet with adequate crude protein (CP). The CP level of negative control (NC) was decreased by 45 and 40 g kg-1 relative to PC for grower and finisher phases, respectively. The subsequent two diets had the same CP levels as NC but with cDDGS added at 50 or 125 g kg-1. The last two diets had the same CP as NC but with CM added at 50 or 100 g kg-1. Dietary CP reduction in corn-SBM diets increased (P < 0.05) the digestibility of Lys (88.5%), Met (90.7%), Thr (77.4%), Cys (80.7%), and Gly (84.7%). Increasing levels of cDDGS linearly decreased (P < 0.05) the digestibility of Asp, Cys, Glu, and Ser, whereas increasing CM level linearly decreased (P < 0.05) the digestibility of Cys, Pro, and Ser. The CP reduction in corn-SBM diets produced downward expression of peptide transporter1 and decreased (P < 0.05) absolute pancreas and ileum weight and length of jejunum and ileum. Partial replacement of SBM with alternative protein feedstuffs (cDDGS or CM) in low-CP diets had minimal effects on AA digestibility and mRNA levels of peptides and AA transporters. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The effect of reducing dietary energy on performance, intestinal morphology and intestinal peptide and amino acid transporters in broiler chicks

AbstractBackgroundPrevious research has investigated the impact of different factors such as gender and age of birds, protein and amino acid levels, protein origin and the physical form of the diet, on the mRNA expression of intestinal peptides and amino acid transporters. Nevertheless, there have been no attempts to examine the impact of dietary energy density on the mRNA abundance of intestinal amino acid and peptide transporters in humans or animals. This is the first reported case in both humans and animals.ObjectivesThis experiment aimed to evaluate the influence of two levels of metabolizable energy ([ME]; 2950 and 2850 kcal ME/kg diet) on performance and jejunal morphology and mRNA expression of amino acid (B0AT, b0,+AT, CAT1 and y+LAT1) and peptide (PepT1) transporters in broiler chickens during the starter period (0–10 days of age).MethodsTwo hundred and seventy day‐old male Ross 308 broiler chickens were randomly assigned to two dietary treatments with nine replicates and fifteen birds in each replicate. At 10 days of age, the outcome variables were measured, which included the assessment of the mRNA abundance of PepT1, b0,+AT, B0AT, y+LAT1 and CAT1 in the jejunal section using real‐time PCR.ResultsThe findings of this study revealed no significant impact of the dietary treatments on feed intake, body weight gain, feed conversion ratio and the jejunal mRNA abundance of PepT1, CAT1 and y+LAT1 (p &gt; 0.05). However, a reduction in dietary ME level from 2950 to 2850 kcal ME/kg diet resulted in a decrease in the mRNA level of B0AT and b0,+AT (p ≤ 0.05). The reduction in dietary energy level did not lead to any significant changes in villus width, villus height, crypt depth, villus surface area and villus height to crypt depth ratio (p &gt; 0.05).ConclusionThe results demonstrated that decreasing dietary energy density may alter the expression of amino acid transporters in the intestine while having no impact on the growth performance and morphometric structure of the intestine.

Differential amino acid transporter expression in adult Drosophila melanogaster tissues.

Organismal macronutrient intake modulates organ and tissue function. Dietary amino acids play essential roles in metabolic processes that support normal tissue growth, repair, and function. For example, in Drosophila melanogaster , protein-deficient diets lead to reduced overall organismal growth during larval development and severely decreased egg production in adult females. Multiple tissues, therefore, must sense and respond to dietary protein input. Amino acid transporter proteins facilitate the movement of amino acids across cellular membranes. Based on high-throughput expression studies, the Drosophila genome is predicted to encode 58 amino acid transporters. We have set out to determine if there are tissue-specific amino acid requirements for proper tissue function by first assessing the complement of amino acid transporters expressed in several adult tissues. Using RT-PCR to assess transcript levels, we find that most of the 24 amino acid transporters examined are expressed in the head, thorax, abdomen, gut, and ovary, while a subset shows differential transcript expression. This work will serve as the foundation for future studies addressing the impact of physiological factors, like nutrition, on amino acid sensing by individual tissues.

Comparative study of susceptibility to methylmercury cytotoxicity in cell types composing rat peripheral nerves: a higher susceptibility of dorsal root ganglion neurons.

Methylmercury is an environmental polluting organometallic compound that exhibits neurotoxicity, as observed in Minamata disease patients. Methylmercury damages peripheral nerves in Minamata patients, causing more damage to sensory nerves than motor nerves. Peripheral nerves are composed of three cell types: dorsal root ganglion (DRG) cells, anterior horn cells (AHCs), and Schwann cells. In this study, we compared cultured these three cell types derived from the rat for susceptibility to methylmercury cytotoxicity, intracellular accumulation of mercury, expression of L-type amino acid transporter 1 (LAT1), which transports methylmercury into cells, and expression of multidrug resistance-associated protein 2 (MRP2), which transports methylmercury-glutathione conjugates into the extracellular space. Of the cells examined, we found that DRG cells were the most susceptible to methylmercury with markedly higher intracellular accumulation of mercury. The constitutive level of LAT1 was higher and that of MRP2 lower in DRG cells compared with those in AHC and Schwann cells. Additionally, decreased cell viability caused by methylmercury was significantly reduced by either the LAT1 inhibitor, JPH203, or siRNA-mediated knockdown of LAT1. On the other hand, an MRP2 inhibitor, MK571, significantly intensified the decrease in the cell viability caused by methylmercury. Our results provide a cellular basis for sensory neve predominant injury in the peripheral nerves of Minamata disease patients.

Metabolic requirements of CD160 expressing memory-like NK cells in Gram-negative bacterial infection.

Unique metabolic requirements accompany the development and functional fates of immune cells. How cellular metabolism is important in natural killer (NK) cells and their memory-like differentiation in bacterial infections remains elusive. Here, we utilise our established NK cell memory assay to investigate the metabolic requirement for memory-like NK cell formation and function in response to the Gram-negative intracellular bacteria Burkholderia pseudomallei (BP), the causative agent of melioidosis. We demonstrate that CD160+ memory-like NK cells upon BP stimulation upregulate glucose and amino acid transporters in a cohort of recovered melioidosis patients which is maintained at least 3-month post-hospital admission. Using an in vitro assay, human BP-specific CD160+ memory-like NK cells show metabolic priming including increased expression of glucose and amino acid transporters with elevated glucose uptake, increased mTOR activation and mitochondrial membrane potential upon BP re-stimulation. Antigen-specific and cytokine-induced IFN-γ production of this memory-like NK cell subset are highly dependent on oxidative phosphorylation (OXPHOS) with some dependency on glycolysis, whereas the formation of CD160+ memory-like NK cells in vitro is dependent on fatty acid oxidation and OXPHOS and further increased by metformin. This study reveals the link between metabolism and cellular function of memory-like NK cells, which can be exploited for vaccine design and for monitoring protection against Gram-negative bacterial infection.

Dietary methionine supplementation during the estrous cycle improves follicular development and estrogen synthesis in rats.

The follicle is an important unit for the synthesis of steroid hormones and the oocyte development and maturation in mammals. However, the effect of methionine supply on follicle development and its regulatory mechanism are still unclear. In the present study, we found that dietary methionine supplementation during the estrous cycle significantly increased the number of embryo implantation sites, as well as serum contents of a variety of amino acids and methionine metabolic enzymes in rats. Additionally, methionine supplementation markedly enhanced the expression of rat ovarian neutral amino acid transporters, DNA methyltransferases (DNMTs), and cystathionine gamma-lyase (CSE); meanwhile, it significantly increased the ovarian concentrations of the metabolite S-adenosylmethionine (SAM) and glutathione (GSH). In vitro data showed that methionine supply promotes rat follicle development through enhancing the expression of critical gene growth differentiation factor 9 and bone morphogenetic protein 15. Furthermore, methionine enhanced the relative protein and mRNA expression of critical genes related to estrogen synthesis, ultimately increasing estrogen synthesis in primary ovarian granulosa cells. Taken together, our results suggested that methionine promoted follicular growth and estrogen synthesis in rats during the estrus cycle, which improved embryo implantation during early pregnancy. These findings provided a potential nutritional strategy to improve the reproductive performance of animals.