An in vivo study of ovine placental transport of essential amino acids

2001 ◽  
Vol 280 (1) ◽  
pp. E31-E39 ◽  
Author(s):  
Cinzia L. Paolini ◽  
Giacomo Meschia ◽  
Paul V. Fennessey ◽  
Adrian W. Pike ◽  
Cecilia Teng ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Concentration ◽  
Essential Amino Acids ◽  
Maternal Plasma ◽  
Transport Systems ◽  
Fetal Plasma ◽  
Placental Transport ◽  
Rate Limiting ◽  
Pulse Flux

Under normal physiological conditions, essential amino acids (EA) are transported from mother to fetus at different rates. The mechanisms underlying these differences include the expression of several amino acid transport systems in the placenta and the regulation of EA concentrations in maternal and fetal plasma. To study the relation of EA transplacental flux to maternal plasma concentration, isotopes of EA were injected into the circulation of pregnant ewes. Measurements of concentration and molar enrichment in maternal and fetal plasma and of umbilical plasma flow were used to calculate the ratio of transplacental pulse flux to maternal concentration (clearance) for each EA. Five EA (Met, Phe, Leu, Ile, and Val) had relatively high and similar clearances and were followed, in order of decreasing clearance, by Trp, Thr, His, and Lys. The five high-clearance EA showed strong correlation ( r 2 = 0.98) between the pulse flux and maternal concentration. The study suggests that five of the nine EA have similar affinity for a rate-limiting placental transport system that mediates rapid flux from mother to fetus, and that differences in transport rates within this group of EA are determined primarily by differences in maternal plasma concentration.

scholarly journals Placental Transport of Leucine, Phenylalanine, Glycine, and Proline in Intrauterine Growth-Restricted Pregnancies

2001 ◽  
Vol 86 (11) ◽  
pp. 5427-5432 ◽  
Author(s):  
Cinzia L. Paolini ◽  
Anna Maria Marconi ◽  
Stefania Ronzoni ◽  
Michela Di Noio ◽  
Paul V. Fennessey ◽  
...  
Keyword(s):  
Amino Acids ◽  
Gestational Age ◽  
Essential Amino Acids ◽  
Enrichment Ratio ◽  
Maternal Circulation ◽  
Intrauterine Growth ◽  
Fetal Blood Sampling ◽  
Placental Transport ◽  
Appropriate For Gestational Age

l-[1-13C]Leucine,[ 1-13C]glycine, l-[1-13C]phenylalanine, and l-[1-13C]proline were infused as a bolus into the maternal circulation of seven appropriate for gestational age at 30.3 ± 3.0 wk and 7 intrauterine growth-restricted pregnancies at 26.5 ± 1.0 wk gestation to investigate placental transport in vivo. Umbilical venous samples were obtained at the time of in utero fetal blood sampling at 450± 74 sec from the bolus injection. In normal pregnancies the fetal/maternal (F/M) enrichment ratios for leucine (0.76 ± 0.06) and phenylalanine (0.77 ± 0.06) were higher (P < 0.01) than the F/M ratios for glycine (0.18 ± 0.04) and proline (0.22 ± 0.02). This suggests that these two essential amino acids rapidly cross the placenta in vivo. Compared with the essentials, both glycine and proline had significantly lower F/M enrichment ratios, which were not different from each other. The results support the hypothesis that amino acids with high affinity for exchange transporters cross the placenta most rapidly. In intrauterine growth-restricted pregnancies, the F/M enrichment ratio was significantly lower (P < 0.01) for l-[1-13C]leucine (0.76 ± 0.06 vs. 0.48 ± 0.07) and for l-[1-13C]phenylalanine (0.77 ± 0.06 vs. 0.46 ± 0.07) compared with appropriate for gestational age pregnancies reflecting impaired transplacental flux. The F/M enrichment ratio did not differ for[ 1-13C]glycine (0.18 ± 0.04 vs. 0.17 ± 0.03), and l-[1-13C]proline (0.22 ± 0.02 vs. 0.18 ± 0.04).

scholarly journals The effects of hyperphenylalaninaemia on the concentrations of aminoacyl-transfer ribonucleic acid in vivo. A mechanism for the inhibition of neural protein synthesis by phenylalanine

1977 ◽  
Vol 162 (3) ◽  
pp. 527-537 ◽  
Author(s):  
J V Hughes ◽  
T C Johnson
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Present Report ◽  
Periodate Oxidation ◽  
Essential Amino Acids ◽  
Acute Administration ◽  
Trna Species ◽  
Intracellular Concentrations ◽  
Rate Limiting

An acute administration of phenylalanine to neonatal animals has been reported to result in large decreases in the intracellular concentrations of several essential amino acids in neural tissue, as well as an inhibition of neural protein synthesis. The present report evaluates the effects of the loss of amino acids on the concentrations of aminoacyl-tRNA in vivo, with the view that an alteration in the concentrations of specific aminoacyl-tRNA molecules could be the rate-limiting step in brain protein metabolism during hyperphenylalaninaemia. tRNA was isolated from saline- and phenylalanine-injected mice 30-45 min after injection, by using a procedure designed to maintain the concentrations of aminoacyl-tRNA present in vivo. Periodate oxidation of the non-acylated tRNA and aminoacylation with radioactively labelled amino acids was used to determine the proportion of tRNA that was present in vivo as aminoacyl-tRNA. Although decreases in the intracellular concentrations of alanine, lysine and leucine were observed after phenylalanine administration, the concentrations of alanyl-tRNA, lysyl-tRNA and leucyl-tRNA actually increased by 15%. Although tryptophan has been suggested to be rate-limiting during hyperphenylalaninaemia, the proportion of tryptophan tRNA that was acylated was maximal in both normal and hyperphenylalaninaemic animals. This unexpected increase in aminoacyl-tRNA concentration is discussed as perhaps a secondary effect resulting from the phenylalanine-induced inhibition of protein synthesis. In contrast, the proportion of methionine tRNA that was acylated in vivo after phenylalanine administration was demonstrated to be decreased by approx. 17%. When the isoaccepting species of methionine tRNA were separated by reverse-phase column chromatography, three species were separated, one of which was demonstrated to be the initiator species, tRNAfMet, by the selective aminoacylation and formylation with Escherichia coli enzymes. After the administration of phenylalanine, the acylation of each of the three methionine tRNA species was decreased, with the initiator species being lowered by 10%. This effect on aminoacylation of tRNAfMet may be the primary step by which phenylalanine affects neural protein synthesis, and this is consistent with previous reports that re-initiation may be inhibited during hyperphenylalaninaemia.

scholarly journals A multi-enzyme cascade for efficient production of d-p-hydroxyphenylglycine from l-tyrosine

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Xu Tan ◽  
Sheng Zhang ◽  
Wei Song ◽  
Jia Liu ◽  
Cong Gao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Enantiomeric Excess ◽  
Efficient Production ◽  
Enzyme Cascade ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rotation Strategy ◽  
Rate Limiting

AbstractIn this study, a four-enzyme cascade pathway was developed and reconstructed in vivo for the production of d-p-hydroxyphenylglycine (D-HPG), a valuable intermediate used to produce β-lactam antibiotics and in fine-chemical synthesis, from l-tyrosine. In this pathway, catalytic conversion of the intermediate 4-hydroxyphenylglyoxalate by meso-diaminopimelate dehydrogenase from Corynebacterium glutamicum (CgDAPDH) was identified as the rate-limiting step, followed by application of a mechanism-guided “conformation rotation” strategy to decrease the hydride-transfer distance d(C6HDAP−C4NNADP) and increase CgDAPDH activity. Introduction of the best variant generated by protein engineering (CgDAPDHBC621/D120S/W144S/I169P with 5.32 ± 0.85 U·mg−1 specific activity) into the designed pathway resulted in a D-HPG titer of 42.69 g/L from 50-g/L l-tyrosine in 24 h, with 92.5% conversion, 71.5% isolated yield, and > 99% enantiomeric excess in a 3-L fermenter. This four-enzyme cascade provides an efficient enzymatic approach for the industrial production of D-HPG from cheap amino acids.

scholarly journals The rate-limiting step of protein synthesis in vivo and in vitro and the distribution of growing peptides between the puromycinlabile and puromycin-non-labile sites on polyribosomes

1971 ◽  
Vol 122 (3) ◽  
pp. 267-276 ◽  
Author(s):  
D. C. N. Earl ◽  
Susan T. Hindley
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Donor Site ◽  
Peptide Bond ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Donor And Acceptor ◽  
Rate Limiting

1. At 3 min after an intravenous injection of radioactive amino acids into the rat, the bulk of radioactivity associated with liver polyribosomes can be interpreted as growing peptides. 2. In an attempt to identify the rate-limiting step of protein synthesis in vivo and in vitro, use was made of the action of puromycin at 0°C, in releasing growing peptides only from the donor site, to study the distribution of growing peptides between the donor and acceptor sites. 3. Evidence is presented that all growing peptides in a population of liver polyribosomes labelled in vivo are similarly distributed between the donor and acceptor sites, and that the proportion released by puromycin is not an artifact of methodology. 4. The proportion released by puromycin is about 50% for both liver and muscle polyribosomes labelled in vivo, suggesting that neither the availability nor binding of aminoacyl-tRNA nor peptide bond synthesis nor translocation can limit the rate of protein synthesis in vivo. Attempts to alter this by starvation, hypophysectomy, growth hormone, alloxan, insulin and partial hepatectomy were unsuccessful. 5. Growing peptides on liver polyribosomes labelled in a cell-free system in vitro or by incubating hemidiaphragms in vitro were largely in the donor site, suggesting that either the availability or binding of aminoacyl-tRNA, or peptide bond synthesis, must be rate limiting in vitro and that the rate-limiting step differs from that in vivo. 6. Neither in vivo nor in the hemidiaphragm system in vitro was a correlation found between the proportion of growing peptides in the donor site and changes in the rate of incorporation of radioactivity into protein. This could indicate that the intracellular concentration of amino acids or aminoacyl-tRNA limits the rate of protein synthesis and that the increased incorporation results from a rise to a higher but still suboptimum concentration.

Glucose and lactate metabolism in vivo in ovine fetus

1980 ◽  
Vol 239 (3) ◽  
pp. E208-E214 ◽  
Author(s):  
R. L. Prior
Keyword(s):  
Specific Activity ◽  
Maternal Plasma ◽  
Fetal Weight ◽  
Plasma Lactate ◽  
Fetal Plasma ◽  
Lactate Turnover ◽  
Ovine Placenta ◽  
Brachial Vein ◽  
Ovine Fetus

The metabolism of glucose and lactate by the ovine fetus (123-128 days of gestation) was studied; a primed, continuous infusion of [2-3H]glucose and [U-14C]lactate into the brachial vein of six fetuses was used. Fetal plasma lactate concentrations averaged 2.12 +/- 0.25 mM and glucose concentrations averaged 9.3 +/- 1.3 mg/100 ml. Total plasma turnover of lactate was 5.22 +/- 0.7 nmol/h and that of glucose was 3.48 +/- 0.63 nmol x h-1 x kg fetal weight-1. Lactate was converted to glucose at a rate of 1.35 +/- 0.64 mmol x h-1 x kg fetal weight-1, which represented 21.6 +/- 6.0% of the lactate turnover. The percentage of glucose coming from lactate was 48.9 +/- 15.2. The specific activity of maternal plasma glucose was less than 4% of the specific activity of glucose observed in fetal plasma. No radioactivity could be detected in maternal plasma lactate. The data show that the ovine fetus or the fetal-placental unit can convert lactate to glucose by days 123-128 of gestation. A general model presented describes carbohydrate metabolism in the ovine placenta and fetus.

scholarly journals Myofibrillar Protein Abundance and Anabolic Signaling in Myotubes Depleted of E1 Alpha Subunit of Branched-Chain Ketoacid Dehydrogenase Complex

2020 ◽  
Vol 4 (Supplement_2) ◽  
pp. 642-642
Author(s):  
Glory Madu ◽  
Olasunkanmi Adegoke
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Protein Content ◽  
Muscle Protein ◽  
Essential Amino Acids ◽  
Myofibrillar Protein ◽  
L6 Myotubes ◽  
Branched Chain ◽  
Ketoacid Dehydrogenase

Abstract Objectives Branched-chain amino acids (BCAAs) are essential amino acids that are crucial for skeletal muscle anabolism. Thus, alterations in their levels are associated with muscle atrophic diseases such as cancer, chronic inflammatory and neurological disorders. Others have linked impairments in BCAA metabolism to the development of insulin resistance and its sequelae. Compared to the effects of theses amino acids, much less is known on how impairment in BCAA catabolism affects skeletal muscle. BCAA catabolism starts with the reversible transamination by the mitochondrial enzyme branched-chain aminotransferase 2 (BCAT2). This is followed by the irreversible carboxylation, catalyzed by branched-chain ketoacid dehydrogenase (BCKD) complex. We have shown that BCAT2 and BCKD are essential for the differentiation of skeletal myoblasts into myotubes. Here, we investigated the effect of depletion of BCAT2 or of E1a subunit of BCKD in differentiated myotubes. Methods On day 4 of differentiation, L6 myotubes were transfected with the following siRNA oligonucleotides: scrambled (control), BCAT2, or E1a subunit of BCKD. Results Forty-eight hours after transfection, compared to control or BCAT2 siRNA group, we observed improved myotube structure in BCKD-depleted cells. BCKD depletion augmented myofibrillar protein levels: myosin heavy chain (MHC, 2-fold) and tropomyosin (4-fold), P < 0.05, n = 3. To further analyze the increase in myofibrillar protein content, we examined signaling through mTORC1 (mechanistic target of rapamycin complex 1), a vital complex necessary for skeletal muscle anabolism. BCKD depletion increased the phosphorylation of mTORC1 upstream activator AKT (52%, P < 0.05, n = 3), and of mTORC1 downstream substrates by 25%-86%, consistent with the increase in myofibrillar proteins. Finally, in myotubes treated with the catabolic cytokine (tumor necrosis factor-a), BCKD depletion tended to increase the abundance of tropomyosin (a myofibrillar protein). Conclusions We showed that depletion of BCKD enhanced myofibrillar protein content and anabolic signaling.  If these data are confirmed in vivo, development of dietary and other interventions that target BCKD abundance or functions may promote muscle protein anabolism in individuals with muscle wasting conditions. Funding Sources MHRC, NSERC York U.

THE STIMULATION OF INSULIN RELEASE BY ESSENTIAL AMINO ACIDS FROM RABBIT PANCREAS IN VITRO

1970 ◽  
Vol 47 (3) ◽  
pp. 347-356 ◽  
Author(s):  
R. D. G. MILNER
Keyword(s):  
Amino Acids ◽  
Insulin Release ◽  
Incubation Medium ◽  
Statistical Significance ◽  
Adenosine Monophosphate ◽  
Essential Amino Acids ◽  
Isoleucine Leucine ◽  
Rabbit Pancreas

SUMMARY Pieces of rabbit pancreas were incubated in vitro in an incubation medium containing no glucose or 1·5 mg. glucose/ml. In each of these conditions the effect on insulin release of each of the essential amino acids at 5 mm concentration was studied. Leucine was the only essential amino acid that stimulated insulin release to a level which reached statistical significance in an incubation medium containing no glucose. In medium containing 1·5 mg. glucose/ml., arginine, isoleucine, leucine and lysine stimulated insulin release and phenylalanine inhibited insulin release. Glucagon, theophylline or dibutyryl cyclic adenosine monophosphate stimulated insulin release significantly in the presence of leucine but not in the presence of arginine. Arginine stimulated insulin release in the presence of leucine. The results of these experiments characterize further the difference in the mechanism of action of leucine and arginine on the pancreatic β-cell and indicate possible explanations for results obtained in other species in vivo.

scholarly journals Effects of maternal iron restriction in the rat on hypoxia-induced gene expression and fetal metabolite levels

2001 ◽  
Vol 85 (2) ◽  
pp. 193-201 ◽  
Author(s):  
Rohan M. Lewis ◽  
Lynwen A. James ◽  
Junlong Zhang ◽  
Christopher D. Byrne ◽  
C. Nicholas Hales
Keyword(s):  
Amino Acids ◽  
Fetal Growth ◽  
Growth Retardation ◽  
Plasma Levels ◽  
Plasma Cholesterol ◽  
Maternal Plasma ◽  
Fetal Growth Retardation ◽  
Restricted Diet ◽  
Fetal Plasma ◽  
Restricted Group

The mechanism by which maternal Fe deficiency in the rat causes fetal growth retardation has not been clearly established. This study compared the effects on the fetuses from dams fed a control diet with two groups of dams fed Fe-restricted diets. One Fe-restricted group was fed the Fe-restricted diet for 1 week prior to mating and throughout gestation and the second Fe-restricted group was fed the Fe-restricted diet for 2 weeks prior to mating and throughout gestation. On day 21 of gestation Fe-restricted dams, and their fetuses, were anaemic. Fetal weight was reduced in both Fe-restricted groups compared with controls. Expression of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) are induced by hypoxia. The levels of HIF-1α mRNA were highest in placenta, then in kidney, heart and liver but were not different between the groups. Levels of plasma VEGF were not different between the groups. Maternal plasma triacylglycerol was decreased in the 1-week Fe-restricted dams compared with controls. Maternal plasma cholesterol and free fatty acid levels were not different between the groups. In fetal plasma, levels of triacylglycerol and cholesterol were decreased in both Fe-restricted groups. In maternal plasma, levels of a number of amino acids were elevated in both Fe-restricted groups. In contrast, levels of a number of amino acids in fetal plasma were lower in both Fe-restricted groups. Fetal plasma lactate was increased in Fe-restricted fetuses but fetal plasma glucose and β-hydroxybutyrate were not affected. These changes in fetal metabolism may contribute to fetal growth retardation in this model. This study does not support the hypothesis that the Fe-restricted fetus is hypoxic.

Leucine as a regulator of whole body and skeletal muscle protein metabolism in humans

1992 ◽  
Vol 263 (5) ◽  
pp. E928-E934 ◽  
Author(s):  
K. S. Nair ◽  
R. G. Schwartz ◽  
S. Welle
Keyword(s):  
Amino Acids ◽  
Protein Degradation ◽  
Protein Metabolism ◽  
Muscle Protein ◽  
Plasma Concentrations ◽  
Essential Amino Acids ◽  
Whole Body ◽  
Skeletal Muscle Protein ◽  
Muscle Protein Metabolism

Leucine has been proposed as an in vivo regulator of protein metabolism, although the evidence for this in humans remains inconclusive. To test this hypothesis, we infused either L-leucine (154 +/- 1 mumol.kg-1 x h-1) or saline intravenously in six healthy men in two separate studies. L-Leucine infusion increased plasma concentrations of leucine and alpha-ketoisocaproate from 112 +/- 6 and 38 +/- 3 mumol/l to 480 +/- 27 (P < 0.001) and 94 +/- 13 mumol/l (P < 0.001), respectively, without any significant change in circulating insulin or C peptide levels. Leucine infusion decreased plasma concentrations of several amino acids and decreased whole body valine flux and valine oxidation (using L-[1-13C]valine as a tracer) and phenylalanine flux (using [2H5]-phenylalanine as a tracer). According to arteriovenous differences across the leg, the net balance of phenylalanine, valine, and lysine shifted toward greater retention during leucine infusion, whereas alanine balance did not change. Valine release and phenylalanine release from the leg (estimated from the dilution of respective tracers) decreased, indicating inhibition of protein degradation by leucine infusion. We conclude that leucine decreases protein degradation in humans and that this decreased protein degradation during leucine infusion contributes to the decrease in plasma essential amino acids. This study suggests a potential role for leucine as a regulator of protein metabolism in humans.

Protein degradation and synthesis measured with multiple amino acid tracers in vivo

1996 ◽  
Vol 271 (4) ◽  
pp. E733-E741 ◽  
Author(s):  
P. Tessari ◽  
R. Barazzoni ◽  
M. Zanetti ◽  
M. Vettore ◽  
S. Normand ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein S ◽  
Essential Amino Acids ◽  
Body Protein ◽  
Amino Acid Availability ◽  
Before And After ◽  
Branched Chain ◽  
Multiple Amino Acid

Whether tracers of different essential amino acids yield the same estimates of body protein turnover is still uncertain. Therefore, we have simultaneously determined leucine (Leu; using [14C]Leu), phenylalanine (Phe; using [13C]Phe), and tyrosine (Tyr; using [2H2]Tyr) rates of appearance (Ra) from proteolysis (PD), as well as Leu and Phe disposal, into protein synthesis (PS) both before and after an anabolic stimulus in healthy volunteers. Protein anabolism was stimulated by insulin plus a branched-chain amino acid-enriched aromatic amino acid-deficient amino acid solution, which increased Leu (from 145 +/- 9 to 266 +/- 10 mumol/l) but decreased Phe (from 57 +/- 2 to 46 +/- 3) and Tyr (from 58.7 +/- 5.5 to 21.0 +/- 2.2) concentrations. Postabsorptive endogenous Leu Ra (2.04 +/- 0.12 mumol.kg-1.min-1), Phe Ra (0.66 +/- 0.03), and Tyr Ra (0.45 +/- 0.06), as well as rates of PS determined with the leucine (1.65 +/- 0.10 mumol.kg-1.min-1) and the phenylalanine tracer (0.57 +/- 0.03), agreed well with the known abundance of these amino acids in body protein(s). After insulin and amino acids, PD was suppressed (P < 0.001) using all tracers. However, although percent suppression of endogenous Leu Ra (-->1.49 +/- 0.10 mumol.kg-1.min-1, 26 +/- 5%) and Phe Ra (-->0.53 +/- 0.02 mumol.kg-1.min-1, -20 +/- 2%) were comparable, endogenous Tyr Ra was suppressed to a larger extent (-->0.23 +/- 0.02 mumol.kg-1.min-1, -46 +/- 3% P = 0.038). PS was stimulated using the Leu (+24 +/- 7%, P < 0.02) but not the Phe (+6 +/- 4%, not significant) data. We conclude that isotopes of different essential amino acid: provide comparable estimates of PD and PS in the postabsorptive state. However, their responses to an anabolic stimulus may differ, possibly depending on exogenous amino acid availability and/or the resulting plasma levels.

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