scholarly journals Development of Biomarkers for Inhibition of SLC6A19 (B0AT1)—A Potential Target to Treat Metabolic Disorders

2018 ◽  
Vol 19 (11) ◽  
pp. 3597 ◽  
Author(s):  
Kiran Javed ◽  
Qi Cheng ◽  
Adam Carroll ◽  
Thy Truong ◽  
Stefan Bröer
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Metabolic Diseases ◽  
Protein Restriction ◽  
Gas Chromatography Mass Spectrometry ◽  
Roc Curve Analysis ◽  
Neutral Amino Acids ◽  
Faecal Samples

Recent studies have established that dietary protein restriction improves metabolic health and glucose homeostasis. SLC6A19 (B0AT1) is the major neutral amino acid transporter in the intestine and carries out the bulk of amino acid absorption from the diet. Mice lacking SLC6A19 show signs of protein restriction, have improved glucose tolerance, and are protected from diet-induced obesity. Pharmacological blockage of this transporter could be used to induce protein restriction and to treat metabolic diseases such as type 2 diabetes. A few novel inhibitors of SLC6A19 have recently been identified using in vitro compound screening, but it remains unclear whether these compounds block the transporter in vivo. To evaluate the efficacy of SLC6A19 inhibitors biomarkers are required that can reliably detect successful inhibition of the transporter in mice. A gas chromatography mass spectrometry (GC-MS)-based untargeted metabolomics approach was used to discriminate global metabolite profiles in plasma, urine and faecal samples from SLC6A19ko and wt mice. Due to inefficient absorption in the intestine and lack of reabsorption in the kidney, significantly elevated amino acids levels were observed in urine and faecal samples. By contrast, a few neutral amino acids were reduced in the plasma of male SLC6A19ko mice as compared to other biological samples. Metabolites of bacterial protein fermentation such as p-cresol glucuronide and 3-indole-propionic acid were more abundant in SLC6A19ko mice, indicating protein malabsorption of dietary amino acids. Consistently, plasma appearance rates of [14C]-labelled neutral amino acids were delayed in SLC6A19ko mice as compared to wt after intra-gastric administration of a mixture of amino acids. Receiver operating characteristic (ROC) curve analysis was used to validate the potential use of these metabolites as biomarkers. These findings provide putative metabolite biomarkers that can be used to detect protein malabsorption and the inhibition of this transporter in intestine and kidney.

Effect of Cycloheximide on In Vitro and In Vivo Protein Synthesis by Certain Tissues of Rats and Pigeons with Special Reference to Muscle

1973 ◽  
Vol 51 (12) ◽  
pp. 933-941 ◽  
Author(s):  
Njanoor Narayanan ◽  
Jacob Eapen
Keyword(s):  
Amino Acids ◽  
Body Weight ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Amino Acid Incorporation ◽  
Contrast Administration ◽  
Acid Incorporation ◽  
Tissue Homogenates

The effect of cycloheximide in vitro and in vivo on the incorporation of labelled amino acids into protein by muscles, liver, kidneys, and brain of rats and pigeons was studied. In vitro incorporation of amino acids into protein by muscle microsomes, myofibrils, and myofibrillar ribosomes was not affected by cycloheximide. In contrast, administration of the antibiotic into intact animals at a concentration of 1 mg/kg body weight resulted in considerable inhibition of amino acid incorporation into protein by muscles, liver, kidneys, and brain. This inhibition was observed in all the subcellular fractions of these tissues during a period of 10–40 min after the administration of the precursor. Tissue homogenates derived from in vivo cycloheximide-treated animals did not show significant alteration in in vitro amino acid incorporation with the exception of brain, which showed a small but significant enhancement.

Engineering an acetyllysine reader with photocrosslinking amino acid for interactome profiling

2021 ◽  
Author(s):  
Babu Sudhamalla ◽  
Anirban Roy ◽  
Soumen Barman ◽  
Jyotirmayee Padhan
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Interactions ◽  
Unnatural Amino Acids ◽  
Protein Protein Interactions ◽  
Site Specific ◽  
Powerful Approach

The site-specific installation of light-activable crosslinker unnatural amino acids offers a powerful approach to trap transient protein-protein interactions both in vitro and in vivo. Herein, we engineer a bromodomain to...

Amino acid fluxes in rat thin limb segments of Henle’s loop during in vitro microperfusion

1999 ◽  
Vol 277 (2) ◽  
pp. F204-F210 ◽  
Author(s):  
Olga H. Brokl ◽  
William H. Dantzler
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Apparent Movement ◽  
Transepithelial Transport ◽  
Acid Transport ◽  
Henle's Loop ◽  
Vasa Recta ◽  
The Difference

Amino acids are apparently recycled between loops of Henle and vasa recta in the rat papilla in vivo. To examine more closely papillary amino acid transport, we measured transepithelial fluxes ofl-[14C]alanine and [14C]taurine in thin limbs of Henle’s loops isolated from rat papilla and perfused in vitro. In descending thin limbs (DTL) in vitro, unidirectional bath-to-lumen fluxes tended to exceed unidirectional lumen-to-bath fluxes for both radiolabeled amino acids, although the difference was statistically significant only for taurine. In ascending thin limbs (ATL) in vitro, unidirectional lumen-to-bath fluxes tended to exceed unidirectional bath-to-lumen fluxes, although the difference was again statistically significant only for taurine. These results are compatible with apparent directional movements of amino acids in vivo. However, none of the unidirectional fluxes was saturable or inhibitable, an observation compatible with apparent reabsorption from the ATL in vivo but not compatible with apparent movement from vasa recta to DTL in vivo. There was no evidence of net active transepithelial transport when concentrations of radiolabeled amino acids were matched on both sides of perfused tubule segments. These data suggest that regulation of amino acid movement in vivo may involve the vasa recta, not the DTL of Henle’s loops. The data also suggest that transepithelial movement of amino acids in thin limbs of Henle’s loop may occur via a paracellular route.

Correlation between osmoregulation and cell volume regulation

1987 ◽  
Vol 252 (4) ◽  
pp. R768-R773
Author(s):  
M. A. Lang
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Osmotic Pressure ◽  
Cell Volume ◽  
Free Amino Acids ◽  
Volume Regulation ◽  
Free Amino ◽  
Cell Volume Regulation

The euryhaline crab, Callinectes sapidus, behaves both as an osmoregulator when equilibrated in salines in the range of 800 mosM and below and an osmoconformer when equilibrated in salines above 800 mosM. There exists a close correlation between osmoregulation seen in the whole animal in vivo and cell volume regulation studied in vitro. Hyperregulation of the hemolymph osmotic pressure and cell volume regulation both occurred in salines at approximately 800 mosM and below. During long-term equilibration of the crabs to a wide range of saline environments, the total concentration of hemolymph amino acids plus taurine remained below 3 mM. During the first 6 h after an acute osmotic stress to the whole animal, the hemolymph osmotic pressure and Na activity gradually decreased, whereas the free amino acids remained below 3 mM. As the hemolymph osmotic pressure decreased below approximately 850 mosM, the amino acid level began to increase to 17-25 mM. This change was primarily due to increases in glycine, proline, taurine, and alanine. The likely source of the increase in hemolymph free amino acids in vivo is the free amino acid loss from muscle cells observed during cell volume regulation in vitro.

Incorporation of C14-amino acids into protein of isolated diaphragms: effect of epinephrine and norepinephrine

1960 ◽  
Vol 198 (1) ◽  
pp. 54-56 ◽  
Author(s):  
Ira G. Wool
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Glucose Concentration ◽  
Muscle Protein ◽  
Effective Concentration ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Minimal Effective Concentration

When diaphragms isolated from normal rats were incubated with a C14-amino acid the addition of epinephrine or norepinephrine decreased incorporation of C14 into muscle protein. The inhibition occurred whether epinephrine was added in vitro or administered in vivo. The minimal effective concentration of epinephrine in vitro was 0.1 µg/ml. When the glucose concentration in the medium was raised to 300 mg % or more the epinephrine induced inhibition of amino acid incorporation into muscle protein was no longer observed.

Early Hematopoietic Progenitors of Dkc1 Hypomorphic Mutant Mice Display Decreased Proliferation Rate and an Impaired Control of Serine/Arginine-Rich Splicing Factor 4 (Srsf4) Translation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2937-2937
Author(s):  
Antonio R Lucena-Araujo ◽  
Bárbara A Santana-Lemos ◽  
Carol H Thome ◽  
Germano A Ferreira ◽  
Davide Ruggero ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ribosomal Rna ◽  
Proliferation Rate ◽  
Splicing Factor ◽  
Differentially Expressed Proteins ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor

Abstract The X-linked form of dyskeratosis congenita (X-DC) is caused by mutations in DKC1, which encodes for dyskerin: a putative pseudouridine synthase that mediate the posttranscriptional modification of ribosomal RNA (rRNA) through the conversion of uridine (U) to pseudouridine (Ψ). Patients with X-DC display defects in the pseudouridylation of ribosomal RNA that leads to translational upregulation of IRES-containing mRNAs and affects the affinity of the ribosome for these mRNAs. Studies in vivo and in vitro suggest that the pseudouridylation of ribosomal RNA is the underlying mechanism responsible for the enhanced susceptibility to cancer in these patients. Ruggero et al. have previously reported (Ruggero et al. Science. 2003 Jan 10;299(5604):259-62) that hypomorphic Dkc1m (Dkc1m) mice present pancytopenia associated with hypocellularity of the bone marrow (BM) and increased susceptibility to cancer, therefore constituting a reliable model to study the effect of impaired ribogenesis on hematopoiesis and oncogenesis. However, the cellular and molecular mechanisms leading to BM failure in X-DC remain unknown. Here, we describe the in vivo analysis of the proliferation rate of hematopoietic progenitors in Dkc1m mice and compare the proteomic profile of hematopoietic progenitors between Dkc1m mice and wild-type (WT) controls. For in vivo proliferation assays, 1mg of bromodeoxyuridine (BrdU) was injected intraperitoneally, every 6 hours during 24 hours, in 16 mice (eight WT and eight Dkc1m), and BM cells were harvested by flushing bone cavity, followed by immunofluorescence staining of incorporated BrdU and flow cytometric analysis. No differences were detected in the number of lineage-negative (Lin−), Sca1-positive, c-kit-negative (LSK−) cells, multipotent precursors (MPP), common myeloid progenitors (CMP), common lymphoid progenitors (CLP) and immature B (B lin) and erythroid (Eryt) cells between Dkc1m and WT mice. Nevertheless, the BrdU incorporation was lower in LSK cells and CMPs from Dkc1m mice (P<0.05), indicating a lower proliferation rate. Using in vitro stable isotope labelling of amino acids (SILAC) hematopoietic progenitor cells were cultured in complete medium containing 10% fetal bovine serum and cytokines (6 ng/ml mIL-3, 10 ng/ml mIL-6 and 100 ng/ml mSCF). Of note, SILAC is one of the most applied approaches for quantitative proteomics, which uses labeled amino acids contain atoms of different isotopes in cell culture. Briefly, one cell population is cultured in unlabeled medium (control), while a second population is grown in medium substituted with a heavy amino acid (usually arginine 13C and/or lysine 15N). After 2-3 weeks culture, murine hematopoietic progenitors were collected and equal amounts of cells from Dkc1m and WT mice (labeled or not with heavy amino acid) were mixed for protein extraction and analyses. Using this approach, we identified about 3,500 differentially expressed proteins; including proteins related to mRNA assembling and splicing, chromatin remodeling, apoptosis and cell cycle arrest. Interestingly, one of the most differentially expressed proteins between WT and Dkc1m mice (WT light /Dkc1m heavy ratio: 18-fold) was the Serine/arginine-rich splicing factor 4(Srsf4); a member of the splicing factor family (SRSF1, SRSF3 and SRSF4) frequently associated with alternative splicing of genes related to hematopoietic progenitor cell differentiation. Altogether, our preliminary results reveal defects in the transcription/translation of specific mRNAs in Dkc1m cells. Additionally, it is conceivable that the down-regulation of Srsf4 protein could be associated with the low proliferative rate in DKC1m mice and explain the impairment of hematopoiesis in X-DC patients. Disclosures No relevant conflicts of interest to declare.

Trans-tegumental absorption of L-alanine and L-leucine by a monogenean, Diclidophora merlangi

Parasitology ◽  
1978 ◽  
Vol 76 (1) ◽  
pp. 29-37 ◽  
Author(s):  
D. W. Halton
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Sea Water ◽  
Low Molecular Weight ◽  
Acid Uptake ◽  
Neutral Amino Acids ◽  
Freeze Dried ◽  
Significant Difference

SummaryAn in vitro investigation has been made of the relative roles of the gut and tegument in the absorption of the neutral amino acids L-alanine and L-leucine by a marine fish-gill parasite, Diclidophora merlangi. The use of ligatures to preclude oral ingestion of trace-labelled medium has proved inadequate, invariably damaging the tegument, as revealed by stereoscan electron microscopy, and resulting in artifactual levels of absorption. Three alternative procedures have given consistently reliable data on the route of entry of low molecular weight substrates. (1) Ultrastructural examination of worms previously incubated in electron-dense cationic tracers has shown that, in vitro, there is no oral intake of sea water. (2) The suspending of worms in trace-labelled medium with the mouth out of the medium and comparing amino acid uptake with that of worms totally immersed in medium has revealed no statistically significant difference in the absorption levels. (3) Application of section (freeze-dried) auto-radiography to detect diffusible isotope has demonstrated directly transtegumental absorption of a neutral amino acid. It is concluded from these experiments that Diclidophora has a tegumental transport system for absorbing certain neutral amino acids, and whilst, clearly, the worm is sanguinivorous and digests blood in a well-developed gut, it may also be capable of supplementing this diet with low molecular weight organic nutrient absorbed directly from sea water via the tegument.

scholarly journals Reciprocal changes in amino acid metabolism in mammary gland and liver of the lactating rat on starvation and refeeding as indicated by the tissue accumulation of α-amino[1-14C]isobutyrate

1990 ◽  
Vol 268 (3) ◽  
pp. 799-802 ◽  
Author(s):  
A E Tedstone ◽  
V Ilic ◽  
D H Williamson
Keyword(s):  
Amino Acids ◽  
Mammary Gland ◽  
Amino Acid ◽  
Amino Acid Metabolism ◽  
Acid Metabolism ◽  
Reciprocal Relationship ◽  
Nutritional State ◽  
Tissue Accumulation

Measurements of the tissue accumulation in vivo and in vitro by hepatocytes and mammary-gland acini of alpha-amino[1-14C]isobutyrate ([1-14C]AIB) were compared in virgin and lactating rats. The results indicate the existence of a reciprocal relationship between mammary gland and liver for AIB accumulation that is dependent on the lactational and the nutritional state of the rat. This suggests that amino acids are preferentially directed to the mammary gland during active lactation.

scholarly journals The herbicidally active compound N-2-(5-chloro-pyridyl) aminomethylene bisphosphonic acid acts by inhibiting both glutamine and aromatic amino acid biosynthesis

2000 ◽  
Vol 27 (7) ◽  
pp. 677 ◽  
Author(s):  
Giuseppe Forlani ◽  
Barbara Lejczak ◽  
Pawel Kafarski
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Active Compound ◽  
Cultured Cells ◽  
Shikimate Pathway ◽  
Glutamate Synthase ◽  
Phenylalanine Level ◽  
Bisphosphonic Acid

The effect of the herbicidally active compound N-2-(5-chloro-pyridyl)aminomethylene bisphosphonic acid (Cl-pyr-AMBPA), previously found in vitro to inhibit the activity of the first enzyme in the shikimate pathway 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, was investigated in vivo on suspension cultured cells of Nicotiana plumbaginifolia Viviani. Amino acid pool measurement showed an actual reduction of tyrosine, tryptophan and phenylalanine level following the addition of the compound to the growth medium. However, an even stronger effect was noticed for other amino acids, mainly glutamine. When the activity of the enzymes involved in the glutamate cycle was measured in the presence of Cl-pyr-AMBPA, glutamate synthase was unaffected, while glutamine synthetase was significantly inhibited. Contrary to the herbicide phosphinothricin, the inhibitor bound reversibly to the enzyme. Kinetic analysis accounted for an inhibition of uncompetitive type with respect to ammonium, glutamate and ATP, withKivalues of 113, 97 and 39 M, respectively. Only the exogenous supply of a mixture of glutamine and aromatic amino acids relieved cell growth inhibition, suggesting that the phytotoxic properties of Cl-pyr-AMBPA are due to inhibition of key enzymes in both the corresponding pathways.

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