Sodium-dependent Neutral Amino Acid Transporter Research Articles

Dual-Targeting Gold Nanoparticles: Simultaneous Decoration with Ligands for Co-Transporters SGLT-1 and B0AT1

Sodium–glucose co-transporter 1 (SGLT1) and sodium-dependent neutral amino acid transporter (B0AT1) are mainly expressed on the membrane of enterocytes, a type of epithelial cell found in the intestines. In addition to their physiological role in the absorption of nutrients, a protective role in the integrity of the intestinal barrier has been established. The natural ligands of SGLT1 (d-glucose) and of B0AT1 (l-glutamine) can trigger a protective anti-inflammatory effect on the intestinal epithelium. The literature suggests the activation of common intracellular pathways upon engagement of the two transporters, whose functional forms are composed of oligomers or clusters. Simultaneous activation of these two co-transporters could lead to a potential multitarget and synergistic anti-inflammatory effect. Therefore, nanoplatforms containing multiple copies of the ligands could represent chemical tools to study the potential simultaneous activation of the two co-transporters. For these reasons, in this study, a set of different gold nanoparticles decorated with derivatives of d-glucose and of l-glutamine were designed and prepared. In particular, the synthesis of suitable sulfur-ending functionalized ligand derivatives, including a C-glucoside derivative, their anchoring to gold nanoparticles and their physical–chemical characterization have been carried out. The obtained nanostructures could represent promising multifunctional platforms for further investigation of the existence of possible multitarget and synergistic effects toward the two co-transporters SGLT1 and B0AT1.

Generation and characterization of a knock-in mouse model for spastic tetraplegia, thin corpus callosum, and progressive microcephaly (SPATCCM).

Solute carrier family 1 member 4(SLC1A4), also referred to as Alanine/Serine/Cysteine/Threonine-preferring Transporter 1(ASCT1), is a sodium-dependent neutral amino acid transporter. It is expressed in many tissues, including the brain, where it is expressed primarily on astrocytes and plays key roles in neuronal differentiation and development, maintaining neurotransmitter homeostasis, and N-methyl-D-aspartate neurotransmission, through regulation of L- and D-serine. Mutations in SLC1A4 are associated with the rare autosomal recessive neurodevelopmental disorder spastic tetraplegia, thin corpus callosum, and progressive microcephaly (SPATCCM, OMIM 616657). Psychomotor development and speech are significantly impaired in these patients, and many develop seizures. We generated and characterized a knock-in mouse model for the most common mutant allele, which results in a single amino acid change (p.Glu256Lys, or E256K). Homozygous mutants had increased D-serine uptake in the brain, microcephaly, and thin corpus callosum and cortex layer 1. While p.E256K homozygotes showed some significant differences in exploratory behavior relative to wildtype mice, their performance in assays for motor coordination, endurance, learning, and memory was normal, and they showed no significant differences in long-term potentiation. Taken together, these results indicate that the impact of the p.E256K mutation on cognition and motor function is minimal in mice, but other aspects of SLC1A4 function in the brain are conserved. Mice homozygous for p.E256K may be a good model for understanding the developmental basis of the corpus callosum and microcephaly phenotypes observed in SPATCCM patients and assessing whether they are rescued by serine supplementation.

Genetic Insights into the Molecular Pathophysiology of Depression in Parkinson's Disease.

Background and Objectives: Parkinson's disease (PD) is a clinically heterogeneous disorder with poorly understood pathological contributing factors. Depression presents one of the most frequent non-motor PD manifestations, and several genetic polymorphisms have been suggested that could affect the depression risk in PD. Therefore, in this review we have collected recent studies addressing the role of genetic factors in the development of depression in PD, aiming to gain insights into its molecular pathobiology and enable the future development of targeted and effective treatment strategies. Materials and Methods: we have searched PubMed and Scopus databases for peer-reviewed research articles published in English (pre-clinical and clinical studies as well as relevant reviews and meta-analyses) investigating the genetic architecture and pathophysiology of PD depression. Results: in particular, polymorphisms in genes related to the serotoninergic pathway (sodium-dependent serotonin transporter gene, SLC6A4, tryptophan hydrolase-2 gene, TPH2), dopamine metabolism and neurotransmission (dopamine receptor D3 gene, DRD3, aldehyde dehydrogenase 2 gene, ALDH2), neurotrophic factors (brain-derived neurotrophic factor gene, BDNF), endocannabinoid system (cannabinoid receptor gene, CNR1), circadian rhythm (thyrotroph embryonic factor gene, TEF), the sodium-dependent neutral amino acid transporter B(0)AT2 gene, SLC6A15), and PARK16 genetic locus were detected as altering susceptibility to depression among PD patients. However, polymorphisms in the dopamine transporter gene (SLC6A3), monoamine oxidase A (MAOA) and B (MAOB) genes, catechol-O-methyltransferase gene (COMT), CRY1, and CRY2 have not been related to PD depression. Conclusions: the specific mechanisms underlying the potential role of genetic diversity in PD depression are still under investigation, however, there is evidence that they may involve neurotransmitter imbalance, mitochondrial impairment, oxidative stress, and neuroinflammation, as well as the dysregulation of neurotrophic factors and their downstream signaling pathways.

Dynamic Evolution of Retroviral Envelope Genes in Egg-Laying Mammalian Genomes.

Independently acquired envelope (env) genes from endogenous retroviruses have contributed to the placental trophoblast cell-cell fusion in therian mammals. Egg-laying mammals (monotremes) are an important sister clade for understanding mammalian placental evolution, but the env genes in their genomes have yet to be investigated. Here, env-derived open reading frames (env-ORFs) encoding more than 400 amino acid lengths were searched in the genomes of two monotremes: platypus and echidna. Only two env-ORFs were present in the platypus genome, whereas 121 env-ORFs were found in the echidna genome. The echidna env-ORFs were phylogenetically classified into seven groups named env-Tac1 to -Tac7. Among them, the env-Tac1 group contained only a single gene, and its amino acid sequence showed high similarity to those of the RD114/simian type D retroviruses. Using the pseudotyped virus assay, we demonstrated that the Env-Tac1 protein utilizes echidna sodium-dependent neutral amino acid transporter type 1 and 2 (ASCT1 and ASCT2) as entry receptors. Moreover, the Env-Tac1 protein caused cell-cell fusion in human 293T cells depending on the expression of ASCT1 and ASCT2. These results illustrate that fusogenic env genes are not restricted to placental mammals, providing insights into the evolution of retroviral genes and the placenta.

Sodium-Dependent Neutral Amino Acid Transporter 2 Can Serve as a Tertiary Carrier for l-Type Amino Acid Transporter 1-Utilizing Prodrugs.

Membrane transporters are the key determinants of the homeostasis of endogenous compounds in the cells and their exposure to drugs. However, the substrate specificities of distinct transporters can overlap. In the present study, the interactions of l-type amino acid transporter 1 (LAT1)-utilizing prodrugs with sodium-coupled neutral amino acid transporter 2 (SNAT2) were explored. The results showed that the cellular uptake of LAT1-utilizing prodrugs into a human breast cancer cell line, MCF-7 cells, was mediated via SNATs as the uptake was increased at higher pH (8.5), decreased in the absence of sodium, and inhibited in the presence of unselective SNAT-inhibitor, (α-(methylamino)isobutyric acid, MeAIB). Moreover, docking the compounds to a SNAT2 homology model (inward-open conformation) and further molecular dynamics simulations and the subsequent trajectory and principal component analyses confirmed the chemical features supporting the interactions of the studied compounds with SNAT2, which was found to be the main SNAT expressed in MCF-7 cells.

Establishment of High Titer Baboon Endogenous Retrovirus Envelope Pseudotyped Lentiviral Vector Production Protocol Focusing on Syncytium Formation and Exfoliation

Introduction Cytokine stimulation of hematopoietic stem cells (HSCs) for the expression of virus entry transporter is inevitable to obtain high transduction efficiency for VSVg envelope pseudotyped lentiviral vectors (VSVg-LVs). However, those stimulation induce apoptosis in HSCs derived from patients affected by DNA repair disorder, the establishment of novel transduction methods has been warranted. Sodium-dependent neutral amino acid transporter type-1 and type-2, which are the entry transporter for Baboon endogenous retrovirus (BaEV-Rless) envelope pseudotyped lentiviral vectors (BaEV-Rless-LVs), are highly expressed on the surface of HSCs in absence of cytokine stimulation. Therefore, BaEV-Rless-LVs could be an effective gene therapy tool to increase transduction in HSC with DNA repair-related mutations. However, production of high titer BaEV-Rless-LVs proved to be challenging compared to VSVg-LVs, as the titer of BaEV-Rless-LVs was one thousandth of VSVg-LVs. 293T cells transfected with helper plasmids, including BaEV-Rless plasmid, showed morphological changes such as syncytium formation and exfoliation, likely impairing cell viability and virus production. Therefore, for clinical applications, improved production of BaEV-Rless-LVs is mandatory. To this end, we modified the protocol to prevent the characteristic morphological changes, syncytium formation and exfoliation, which could be the causes of low virus titer. Methods In the attempt to increase viral production, we modified the VSVg-LVs "basic protocol" modified to produce BaEV-Rless-LVs: co-transfection of 0.44 µg of BaEV-Rless envelope plasmid, 1.464 µg of the self-inactivating HIV-1 derived vector encoding green fluorescent protein (GFP) under the control of spleen focus foamy virus promoter (SIN-HIV-SFFV-GFP), 0.336 μg of Rev regulatory plasmid and 0.74 μg of RRE plasmid to 293T cells per well of 6-well-plate, respectively. Virus titration was performed on 293T cells using serial dilution of the viral preparations and assessing 293T GFP+ cells using flow cytometry analysis. To investigate in detail the time-course of syncytium formation and exfoliation of transfected 293T cells, we used by time-lapse analysis to assess critical culture conditions and to guide us on how to further modify the protocol. Results Time-lapse analysis revealed that syncytium formation initiated at 8-hours post-transfection of the helper plasmids and terminated at 18-hours post-transfection. (Figure 1). Exfoliation started at 20-hours post-transfection, soon after completion of syncytium formation. As we hypothesized that the syncytium formation and exfoliation might be the cause of the low viral vector production, we modified three points of the basic protocol: the amount of BaEV-Rless plasmid, harvest timing of virus supernatant and use of poly-l-lysine (PLL) coated plate to avoid syncytium formation and exfoliation of 293T cells. First, to clarify whether the amount of BaEV-Rless plasmid was proportional to syncytium formation and virus titer, we changed the amounts of BaEV-Rless plasmid and compared titers. Interestingly, increasing 1.2-fold of the amount of plasmid raised the titer 3-fold despite syncytium formation. In contrast, even though smaller amounts of BaEV-Rless-plasmid did not induce syncytium formation and exfoliation, the titer of BaEV-Rless-LVs was reduced and/or undetectable. Next, we identified the optimal harvest time for viral titer. Harvest of viral supernatant at 48-hours post-transfection was associated with the highest titer despite completion of syncytium formation and exfoliation of 293T cells. Finally, we compared the titers with or without using PLL coated plate. Using PLL coated plate prevented exfoliation of transfected 293T cells and doubled the titer. Eventually, combination of using 1.2-folds of BaEV-Rless-plasmid, harvest of virus supernatant at 48-hours post-transfection and using PLL coated plate resulted in a 10-fold titer increase compared with the basic protocol (Figure 2). Conclusion Our novel high titer protocol for BaEV-Rless-LVs results in 10-fold titer increase compared to the basic protocol, and it could be utilized to develop clinical applications for DNA repair-related disorders. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Identification and characterization of a novel SNAT2 (SLC38A2) inhibitor reveals synergy with glucose transport inhibition in cancer cells.

SNAT2 (SLC38A2) is a sodium-dependent neutral amino acid transporter, which is important for the accumulation of amino acids as nutrients, the maintenance of cellular osmolarity, and the activation of mTORC1. It also provides net glutamine for glutaminolysis and consequently presents as a potential target to treat cancer. A high-throughput screening assay was developed to identify new inhibitors of SNAT2 making use of the inducible nature of SNAT2 and its electrogenic mechanism. Using an optimized FLIPR membrane potential (FMP) assay, a curated scaffold library of 33934 compounds was screened to identify 3-(N-methyl (4-methylphenyl)sulfonamido)-N-(2-trifluoromethylbenzyl)thiophene-2-carboxamide as a potent inhibitor of SNAT2. In two different assays an IC50 of 0.8–3 µM was determined. The compound discriminated against the close transporter homologue SNAT1. MDA-MB-231 breast cancer and HPAFII pancreatic cancer cell lines tolerated the SNAT2 inhibitor up to a concentration of 100 µM but in combination with tolerable doses of the glucose transport inhibitor Bay-876, proliferative growth of both cell lines was halted. This points to synergy between inhibition of glycolysis and glutaminolysis in cancer cells.

Effects of Total Sulfur Amino Acids on Growth Performance, Immunity, and Meat Yield in Broilers Fed Diets With and Without Antibiotics.

Two experiments were conducted to evaluate the effects of digestible sulfur amino acids (SAA) on performance, carcass yield, immunity, and amino acid transporters in broilers fed diets with or without an antibiotic growth promoter (AGP). In experiment 1, a total of 250 1-day-old Cobb500 male chicks were assigned to battery cages with two levels of AGP (0 and 0.05% bacitracin) and five levels of SAA (0.7, 0.8, 0.9, 1.0, and 1.1%) for 21 d. In experiment 2, a total of 900 1-day-old Cobb500 male chicks were assigned to floor pens with two levels of AGP and three levels of SAA for the starter (0.7, 0.8, and 0.9%) or finisher phase (0.52, 0.62, and 0.72%) for 42 d. In experiment 1, from 0 to 7 d, the body weight gain (BWG) was the lowest for birds fed 0.7% SAA. The AGP significantly decreased the feed conversion ratio (FCR), and birds fed 0.9 and 1.1% SAA had significantly lower FCR than 0.7% SAA. From 8 to 14 d, for the AGP-fed birds, the lowest BWG was observed in the 0.7% SAA group. In birds not fed AGP, birds fed 0.8% SAA had higher BWG than 0.7 and 1.1% SAA. Birds fed 0.7% SAA diet had lower feed intake (FI) than 0.8% SAA and higher FCR than 0.8, 0.9, and 1.0% SAA. In experiment 2, from 0 to 21 d, the lowest BWG and the highest FCR were observed in birds fed 0.7% SAA, whereas birds fed 0.9% SAA had the highest BWG and lowest FCR. From 22 to 42 d, FCR was lower for birds fed AGP, and for birds fed 0.72%. Interactions between the factors were found for FI and BWG. The whole thigh and wing weights were the highest for 0.62% SAA, and the pectoralis major weight was higher for birds fed 0.62% SAA than those fed 0.52% SAA. There was an interaction between SAA and AGP for Lat1 (large neutral amino acid transporter) expression, and AGP-fed birds had higher expression of ileal interleukin 1β (Il−1β gene). The interleukin 10 (Il-10) expression was upregulated in the ileum. There was an interaction between factors for sodium-dependent neutral amino acid transporter B [0] AT1 (SLC6A19) expression. The results suggested that both AGP and SAA supplementation would affect the growth performance of the broilers.

LAT1 and SNAT2 Protein Expression and Membrane Localization of LAT1 Are Not Acutely Altered by Dietary Amino Acids or Resistance Exercise Nor Positively Associated with Leucine or Phenylalanine Incorporation in Human Skeletal Muscle.

The influx of essential amino acids into skeletal muscle is primarily mediated by the large neutral amino acid transporter 1 (LAT1), which is dependent on the glutamine gradient generated by the sodium-dependent neutral amino acid transporter 2 (SNAT2). The protein expression and membrane localization of LAT1 may be influenced by amino acid ingestion and/or resistance exercise, although its acute influence on dietary amino acid incorporation into skeletal muscle protein has not been investigated. In a group design, healthy males consumed a mixed carbohydrate (0.75 g·kg−1) crystalline amino acid (0.25 g·kg−1) beverage enriched to 25% and 30% with LAT1 substrates L-[1-13C]leucine (LEU) and L-[ring-2H5]phenylalanine (PHE), respectively, at rest (FED: n = 7, 23 ± 5 y, 77 ± 4 kg) or after a bout of resistance exercise (EXFED: n = 7, 22 ± 2 y, 78 ± 11 kg). Postprandial muscle biopsies were collected at 0, 120, and 300 min to measure transporter protein expression (immunoblot), LAT1 membrane localization (immunofluorescence), and dietary amino acid incorporation into myofibrillar protein (ΔLEU and ΔPHE). Basal LAT1 and SNAT2 protein contents were correlated with each other (r = 0.55, p = 0.04) but their expression did not change across time in FED or EXFED (all, p > 0.05). Membrane localization of LAT1 did not change across time in FED or EXFED whether measured as outer 1.5 µm intensity or membrane-to-fiber ratio (all, p > 0.05). Basal SNAT2 protein expression was not correlated with ΔLEU or ΔPHE (all, p ≥ 0.05) whereas basal LAT1 expression was negatively correlated with ΔPHE in FED (r = −0.76, p = 0.04) and EXFED (r = −0.81, p = 0.03) but not ΔLEU (p > 0.05). Basal LAT1 membrane localization was not correlated with ΔLEU or ΔPHE (all, p > 0.05). Our results suggest that LAT1/SNAT2 protein expression and LAT1 membrane localization are not influenced by acute anabolic stimuli and do not positively influence the incorporation of dietary amino acids for de novo myofibrillar protein synthesis in healthy young males.

Birth Weight and Nutrient Restriction Affect Jejunal Enzyme Activity and Gene Markers for Nutrient Transport and Intestinal Function in Piglets.

Simple SummaryBirth weight and nutrient utilization are thought to have significant effects on intestinal development in neonatal pigs. The present study evaluated the impact of low and normal birth weight with and without nutrient restriction during the neonatal period on jejunal development. The results observed suggest that during the first 28 d of life, birth weight had greater effects on intestinal development than nutrient level, however, at d 56, the nutrient level was a significant contributor to intestinal function and enzyme activity compared to birth weight. Taken together, both birth weight and nutrient restriction have effects on intestinal development, but may have a greater impact in early life (d 28).Significant variation in the birth weight of piglets has arisen due to increased sow prolificacy. Intestinal development and function may be affected by birth weight. Low birth weight (LBW) pigs may also have reduced feed intake, leading to further impairment of intestinal development. The objective of this study was to examine the intestinal development pattern of LBW and normal birth weight (NBW) piglets with normal nutrition (NN) or restricted nutrition (RN) in the pre-weaning period. Jejunal intestinal samples were analyzed for target gene expression and enzyme activity at d 28 (weaning) and d 56 (post-weaning). At d 28, excitatory amino acid transporter (EAAC1) and sodium-dependent neutral amino acid transporter (B0AT1) were downregulated in LBW compared to NBW pigs (p < 0.05). On d 56, B0AT1 and ASCT2 (glutamine transporter) were downregulated in RN compared to NN pigs (p < 0.05), regardless of birth weight. Peptide transporter 1 (PepT1) expression was downregulated in LBW compared to NBW pigs at 28 d (p < 0.05), with no effects of treatments at 56 d. Sodium-glucose transporter-1 (SGLT1) was upregulated in NBW-NN compared to LBW-NN pigs (p < 0.05) at 28 d. Alkaline phosphatase (ALP) was upregulated in LBW-RN at d 28. At d 56, claudin-3 (CLDN-3) and Zonular occludin-1 (ZO-1) were upregulated in NN compared to RN pigs (p < 0.05). There were no treatment effects on ALP, maltase, or sucrase activity at 28 d. However, at 56 d, ALP was upregulated in NBW-NN pigs while sucrase activity was upregulated in NN pigs (p < 0.05). The results demonstrate differences in jejunal gene expression associated with birth weight, with reduced gene expression of amino acid transporters (PepT1, EAAC1, B0AT1) in LBW compared to NBW pigs (p < 0.05). While neonatal nutrient restriction had minimal effects at 28 d and d 56 for tight junction protein transcript abundance, neutral amino acid transporter abundance was upregulated in NN pigs compared to RN piglets (p < 0.05).

Rational design of ASCT2 inhibitors using an integrated experimental-computational approach

ASCT2 (SLC1A5) is a sodium-dependent neutral amino acid transporter that controls amino acid homeostasis in peripheral tissues. In cancer, ASCT2 is up-regulated where it modulates intracellular glutamine levels, fueling cell proliferation. Nutrient deprivation via ASCT2 inhibition provides a potential strategy for cancer therapy. Here, we rationally designed stereospecific inhibitors exploiting specific subpockets in the substrate binding site using computational modeling and cryo-electron microscopy (cryo-EM). The final structures combined with molecular dynamics simulations reveal multiple pharmacologically relevant conformations in the ASCT2 binding site as well as a previously unknown mechanism of stereospecific inhibition. Furthermore, this integrated analysis guided the design of a series of unique ASCT2 inhibitors. Our results provide a framework for future development of cancer therapeutics targeting nutrient transport via ASCT2, as well as demonstrate the utility of combining computational modeling and cryo-EM for solute carrier ligand discovery.

The Amino Acid-mTORC1 Pathway Mediates APEC TW-XM-Induced Inflammation in bEnd.3 Cells.

The blood–brain barrier (BBB) is key to establishing and maintaining homeostasis in the central nervous system (CNS); meningitis bacterial infection can disrupt the integrity of BBB by inducing an inflammatory response. The changes in the cerebral uptake of amino acids may contribute to inflammatory response during infection and were accompanied by high expression of amino acid transporters leading to increased amino acid uptake. However, it is unclear whether amino acid uptake is changed and how to affect inflammatory responses in mouse brain microvascular endothelial (bEnd.3) cells in response to Avian Pathogenic Escherichia coli TW-XM (APEC XM) infection. Here, we firstly found that APEC XM infection could induce serine (Ser) and glutamate (Glu) transport from extracellular into intracellular in bEnd.3 cells. Meanwhile, we also shown that the expression sodium-dependent neutral amino acid transporter 2 (SNAT2) for Ser and excitatory amino acid transporter 4 (EAAT4) for Glu was also significantly elevated during infection. Then, in amino acid deficiency or supplementation medium, we found that Ser or Glu transport were involving in increasing SNAT2 or EAAT4 expression, mTORC1 (mechanistic target of rapamycin complex 1) activation and inflammation, respectively. Of note, Ser or Glu transport were inhibited after SNAT2 silencing or EAAT4 silencing, resulting in inhibition of mTORC1 pathway activation, and inflammation compared with the APEC XM infection group. Moreover, pEGFP-SNAT2 overexpression and pEGFP-EAAT4 overexpression in bEnd.3 cells all could promote amino acid uptake, activation of the mTORC1 pathway and inflammation during infection. We further found mTORC1 silencing could inhibit inflammation, the expression of SNAT2 and EAAT4, and amino acid uptake. Taken together, our results demonstrated that APEC TW-XM infection can induce Ser or Glu uptake depending on amino acid transporters transportation, and then activate amino acid-mTORC1 pathway to induce inflammation in bEnd.3 cells.

Amino Acid Carriers of the Solute Carrier Families 7 (SLC7) and 38 (SLC38) Are Involved in Leucine Sensing in the Brain of Atlantic Salmon (Salmo salar)

Sensing of amino acids in fish brain, especially branched-chain amino acids (BCAA) like leucine, is involved in regulation of feed intake through different mechanisms. However, there is limited information regarding the possible involvement of mechanisms dependent on amino acid carriers of the solute carrier families (SLC) known to be key regulators of intracellular leucine concentration, namely L-type amino acid transporter 1 (LAT1), and sodium-dependent neutral amino acid transporter 2 (SNAT2) and 9,(SNAT9), for which evidence of their participation is available in mammals. Comparative analysis amongst sequences revealed a complex pattern of paralogues in Atlantic salmon, for LAT1 (slc7a5aa, slc7a5ab, slc7a5ba, slc7a5bb, slc7a5ca, and slc7a5cb), SNAT2 (slc38a2a and slc38a2b) and SNAT9 (slc38a9). After establishing phylogenetic relationships of the different paralogues evaluated, samples of the selected brain areas were taken from Atlantic salmon to assess tissue distribution of transcripts. In an additional experiment, fish were fed two diets with different levels of leucine (high leucine: 35 g/kg vs. control leucine: 27.3 g/kg). The high leucine diet resulted in lower feed intake and increased mRNA abundance of specific paralogues of LAT1 (slc7a5aa, slc7a5ab, and slc7a5bb) and SNAT2 (slc38a2a and slc38a2b) though apparently not for SNAT9 in brain areas like hypothalamus and telencephalon involved in food intake regulation. The results obtained suggest a role for members of the SLC family in the anorectic effect of leucine and thus their involvement as additional amino acid sensing mechanism not characterised so far in fish regulation of feed intake.

B0AT1 Amino Acid Transporter Complexed With SARS-CoV-2 Receptor ACE2 Forms a Heterodimer Functional Unit: In Situ Conformation Using Radiation Inactivation Analysis.

The SARS-CoV-2 receptor, angiotensin-converting enzyme-2 (ACE2), is expressed at levels of greatest magnitude in the small intestine as compared with all other human tissues. Enterocyte ACE2 is coexpressed as the apical membrane trafficking partner obligatory for expression and activity of the B0AT1 sodium-dependent neutral amino acid transporter. These components are assembled as an [ACE2:B0AT1]2 dimer-of-heterodimers quaternary complex that putatively steers SARS-CoV-2 tropism in the gastrointestinal (GI) tract. GI clinical symptomology is reported in about half of COVID-19 patients, and can be accompanied by gut shedding of virion particles. We hypothesized that within this 4-mer structural complex, each [ACE2:B0AT1] heterodimer pair constitutes a physiological “functional unit.” This was confirmed experimentally by employing purified lyophilized enterocyte brush border membrane vesicles exposed to increasing doses of high-energy electron radiation from a 16 MeV linear accelerator. Based on radiation target theory, the results indicated the presence of Na+-dependent neutral amino acid influx transport activity functional unit with target size molecular weight 183.7 ± 16.8 kDa in situ in intact apical membranes. Each thermodynamically stabilized [ACE2:B0AT1] heterodimer functional unit manifests the transport activity within the whole ∼345 kDa [ACE2:B0AT1]2 dimer-of-heterodimers quaternary structural complex. The results are consistent with our prior molecular docking modeling and gut–lung axis approaches to understanding COVID-19. These findings advance understanding the physiology of B0AT1 interaction with ACE2 in the gut, and thereby contribute to translational developments designed to treat or mitigate COVID-19 variant outbreaks and/or GI symptom persistence in long-haul postacute sequelae of SARS-CoV-2.

SLC6A15 acts as a tumor suppressor to inhibit migration and invasion in human papillary thyroid cancer.

Solute Carrier Family 6 Member 15 (SLC6A15), a sodium-dependent neutral amino acid transporter, has been found with dysregulated expression in several kinds of cancers. However, the expression pattern and the biological functions of SLC6A15 in papillary thyroid cancer (PTC) remain unknown. In this study, we found that SLC6A15 was downregulated in PTC, which was related to N classification. Ectopic overexpression of SLC6A15 impaired migratory and invasive abilities of PTC cell in vitro. In addition, we identified intercellular adhesion molecule-1, a vital oncogene in thyroid cancer progression, was involved in the effects of SLC6A15 on PTC cell. These results indicate that SLC6A15 acts as a tumor suppressor and might be a potential therapeutic target in the treatment of PTC.

Dietary Insect Powder Protein Sources Improve Protein Utilization by Regulation on Intestinal Amino Acid-Chemosensing System.

Simple SummaryInsect powders, including Tenebrio molitor (TM), Musca domestica larvae (MDL) and Zophobas morio (ZM), as high-quality and renewable protein sources are commonly applied in livestock and poultry feed production. The molecular effect of insect protein on amino acid metabolism in pigs needs to be explored. We found that insect powder as a protein source in feed regulated the mTOR signal pathway and improved amino acid transportation in the intestine for growth promotion. Insect powder may be a potentially promising protein source for pig production.This study was conducted to evaluate the effects of dietary insect powder supplementation as a protein source on plasma amino acid profiles, intestinal amino acid transport and sensing in a piglet model. A total of 144 weanling piglets were randomly assigned to four experimental diets for two phases (Days 1–28 and Days 29–56), to assess the effects on amino acid profiles and transportation in the segments of the intestine. The groups were basal diet (control), control diet plus Tenebrio molitor (TM), control diet plus Musca domestica larvae (MDL) and control diet plus Zophobas morio (ZM). The plasma free amino acid levels were stable comparable among treatments, except that the lysine level was significantly reduced by dietary MDL and ZM supplementation in the first phase (p < 0.05). In the 1st phase, the sensitivity of intestinal segments to the regulation of the amino acid level by insect powder supplementation follows sequence: colon > ileum > jejunum, while the order switched to jejunum > colon > ileum in the 2nd phase. The relative RNA expressions of mitogen-activated protein 4 kinase 3 (MAP4K3), sodium dependent neutral amino acid transporter2 (SNAT2), the transient receptor potential cation channel subfamily V member 1 (TRPV1) and taste 1 receptor member 1/3 (T1R3) in the segments of the intestine were affected by different dietary insect powder supplementation. G protein-coupled receptor family C group 6 member A (GPRC6A) level in the jejunal and colonic mucosa was upregulated by MDL supplementation (p < 0.05). These results indicated that dietary insects improved the metabolism of the amino acid in the prophase (the 1st phase) through regulating the sensing gene and mTOR signal pathway in intestinal mucosa by targeting different receptors. The finding demonstrates that the insect powder is a potentially promising source for protein deposition.

L-carnitine increases cell proliferation and amino acid transporter expression via the activation of insulin-like growth factor I signaling pathway in rat trophoblast cells.

Early embryo implantation and development is primarily determined by the homeostasis between cellular apoptosis and proliferation as well as placental nutrient transporters. Recent studies showed that L‐carnitine enhances female reproductive performance. However, the potential function of L‐carnitine on placenta is largely unknown. In our study, primary rat trophoblast cells were separated and cultured for 12 hr in medium containing various concentrations of L‐carnitine (0, 1, 10, and 50 mM). Placenta trophoblast cells treated with 50 mM L‐carnitine increased the proportion of cells in S phase of the cell cycle (p < .05). In addition, live cell percentage was increased when treated with either 10 mM or 50 mM L‐carnitine, which was accompanied with decreased necrotic cells, late apoptotic cells, and early apoptotic cells (p < .05). Compared with the control treatment, the mRNA expression of insulin‐like growth factor I (IGF‐1) and insulin‐like growth factor I receptor (IGF‐1R) was higher in rat placenta trophoblasts treated with either 10 mM or 50 mM L‐carnitine (p < .05). Similarly, sodium‐dependent neutral amino acid transporter (SNAT)‐1 and SNAT2 were up‐regulated in both mRNA and protein levels when trophoblast cells were treated with 50 mM L‐carnitine (p < .05). Inhibiting downstream targets (Akt or ERK signaling pathways) of IGF‐1 signaling pathway partially blocked the effect the L‐carnitine‐induced increase in protein abundances of SNAT1 and SNAT2. Collectively, our data showed protective role of L‐carnitine on placenta trophoblast cells through the involvement of IGF‐1 signaling pathway.

Two distinct electrophysiological mechanisms underlie extensive depolarization elicited by 2,4 diaminobutyric acid in leech Retzius neurons

Recent studies suggest that 2,4-DABA, a neurotoxic excitatory amino acid present in virtually all environments, but predominantly in aquatic ecosystems may be a risk factor for development of neurodegenerative diseases in animals and humans. Despite its neurotoxicity and potential environmental importance, mechanisms underlying the excitatory and putative excitotoxic action of 2,4-DABA in neurons are still unexplored. We previously reported on extensive two-stage membrane depolarization and functional disturbances in leech Retzius neurons induced by 2,4-DABA. Current study presents the first detailed look into the electrophysiological processes leading to this depolarization. Intracellular recordings were performed on Retzius neurons of the leech Haemopis sanguisuga using glass microelectrodes and input membrane resistance (IMR) was measured by injecting hyperpolarizing current pulses through these electrodes. Results show that the excitatory effect 2,4-DABA elicits on neurons’ membrane potential is dependent on sodium ions. Depolarizing effect of 5·10−3 mol/L 2,4-DABA in sodium-free solution was significantly diminished by 91% reducing it to 3.26 ± 0.62 mV and its two-stage nature was abrogated. In addition to being sodium-dependent, the depolarization of membrane potential induced by this amino acid is coupled with an increase of membrane permeability, as 2,4-DABA decreases IMR by 8.27 ± 1.47 MΩ (67.60%). Since present results highlight the role of sodium ions, we investigated the role of two putative sodium-dependent mechanisms in 2,4-DABA-induced excitatory effect – activation of ionotropic glutamate receptors and the electrogenic transporter for neutral amino acids. Excitatory effect of 5·10−3 mol/L 2,4-DABA was partially blocked by 10-5 mol/L 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) a non-NMDA receptor antagonist as the first stage of membrane depolarization was significantly reduced by 2.59 ± 0.98 mV (40%), whilst second stage remained unaltered. Moreover, involvement of the sodium-dependent transport system for neutral amino acids was investigated by equimolar co-application of 5·10−3 mol/L 2,4-DABA and L-alanine, a competitive inhibitor of this transporter. Although L-alanine exhibited no effect on the first stage of membrane depolarization elicited by 2,4-DABA, it substantially reduced the second stage (the overall membrane depolarization) from 39.63 ± 2.22 mV to 16.28 ± 2.58 mV, by 58.92%. We therefore propose that the electrophysiological effect of 2,4-DABA on Retzius neurons is mediated by two distinct mechanisms, i.e. by activation of ionotropic glutamate receptor that initiates the first stage of membrane depolarization followed by the stimulation of an electrogenic sodium-dependent neutral amino acid transporter, leading to additional influx of positive charge into the cell and the second stage of depolarization.

Microarray analysis reveals the inhibition of intestinal expression of nutrient transporters in piglets infected with porcine epidemic diarrhea virus

Porcine epidemic diarrhea virus (PEDV) infection can induce intestinal dysfunction, resulting in severe diarrhea and even death, but the mode of action underlying these viral effects remains unclear. This study determined the effects of PEDV infection on intestinal absorption and the expression of genes for nutrient transporters via biochemical tests and microarray analysis. Sixteen 7-day-old healthy piglets fed a milk replacer were randomly allocated to one of two groups. After 5-day adaption, piglets (n = 8/group) were orally administrated with either sterile saline or PEDV (the strain from Yunnan province) at 104.5 TCID50 (50% tissue culture infectious dose) per pig. All pigs were orally infused D-xylose (0.1 g/kg BW) on day 5 post PEDV or saline administration. One hour later, jugular vein blood samples as well as intestinal samples were collected for further analysis. In comparison with the control group, PEDV infection increased diarrhea incidence, blood diamine oxidase activity, and iFABP level, while reducing growth and plasma D-xylose concentration in piglets. Moreover, PEDV infection altered plasma and jejunal amino acid profiles, and decreased the expression of aquaporins and amino acid transporters (L-type amino acid transporter 1, sodium-independent amino acid transporter, B(°,+)-type amino acid transport protein, sodium-dependent neutral amino acid transporter 1, sodium-dependent glutamate/aspartate transporter 3, and peptide transporter (1), lipid transport and metabolism-related genes (lipoprotein lipase, apolipoprotein A1, apolipoprotein A4, apolipoprotein C2, solute carrier family 27 member 2, solute carrier family 27 member 4, fatty acid synthase, and long-chain acyl-CoA synthetase (3), and glucose transport genes (glucose transporter-2 and insulin receptor) in the jejunum. However, PEDV administration increased mRNA levels for phosphoenolpyruvate carboxykinase 1, argininosuccinate synthase 1, sodium/glucose co-transporter-1, and cystic fibrosis transmembrane conductance regulator in the jejunum. Collectively, these comprehensive results indicate that PEDV infection induces intestinal injury and inhibits the expression of genes encoding for nutrient transporters.

Comparative analysis of the liver transcriptome in the red-eared slider Trachemys scripta elegans under chronic salinity stress.

The red-eared slider (Trachemys scripta elegans), identified as one of the 100 most invasive species in the world, is a freshwater turtle originally from the eastern United States and northeastern Mexico. Field investigations have shown that T. s. elegans can survive and lay eggs in saline habitats. In order to understand the molecular mechanisms of salinity adaptation, high-throughput RNA-Seq was utilized to identify the changes in gene expression profiles in the liver of T. s. elegans in response to elevated salinity. We exposed individuals to 0, 5, or 15 psu (practical salinity units) for 30 days. A total of 157.21 million reads were obtained and assembled into 205138 unigenes with an average length of 620 bp and N50 of 964 bp. Of these, 1019 DEGs (differentially expressed genes) were found in the comparison of 0 vs. 5 psu, 1194 DEGs in 0 vs. 15 psu and 1180 DEGs in 5 vs. 15 psu, which are mainly related to macromolecule metabolic process, ion transport, oxidoreductase activity and generation of precursor metabolites and energy by GO (Gene Ontology) enrichment analyses. T. s. elegans can adapt itself into salinity by balancing the entry of sodium and chloride ions via the up-regulation expression genes of ion transport (potassium voltage-gated channel subfamily H member 5, KCNH5; erine/threonine-protein kinase 32, STK32; salt-inducible kinase 1, SIK1; adiponectin, ACDC), and by accumulating plasma urea and free amino acid via the up-regulation expression genes of amino acid metabolism (ornithine decarboxylase antizyme 3, OAZ3; glutamine synthetase, GLUL; asparaginase-like protein 1b, ASRGL; L-amino-acid oxidase-like, LAAO; sodium-dependent neutral amino acid transporter B, SLC6A15s; amino acid permease, SLC7A9) in response to osmotic regulation. An investment of energy to maintain their homeostatic balance is required to salinity adaptation, therefore, the genes related to energy production and conversion (F-ATPase protein 6, ATP6; cytochrome c oxidase subunit I, COX1; cytochrome c oxidase subunit III, COX3; cytochrome b, CYTb; cytochrome P450 17A1, CYP17A1) were up-regulated with the increase of gene expression associated with lipid metabolism (apolipoprotein E precursor, APoE; coenzyme Q-binding protein, CoQ10; high-density lipoprotein particle, SAA) and carbohydrate metabolism (HK, MIP). These findings improve our understanding of the underlying molecular mechanisms involved in salinity adaptation and provide general guidance to illuminate the invasion potential of T. s. elegans into saline environments.