Acute supplementation of amino acids increases net protein accretion in IUGR fetal sheep

Placental insufficiency decreases fetal amino acid uptake from the placenta, plasma insulin concentrations, and protein accretion, thus compromising normal fetal growth trajectory. We tested whether acute supplementation of amino acids or insulin into the fetus with intrauterine growth restriction (IUGR) would increase net fetal protein accretion rates. Late-gestation IUGR and control (CON) fetal sheep received acute, 3-h infusions of amino acids (with euinsulinemia), insulin (with euglycemia and euaminoacidemia), or saline. Fetal leucine metabolism was measured under steady-state conditions followed by a fetal muscle biopsy to quantify insulin signaling. In CON, increasing amino acid delivery rates to the fetus by 100% increased leucine oxidation rates by 100%. In IUGR, amino acid infusion completely suppressed fetal protein breakdown rates but increased leucine oxidation rate by only 25%, resulting in increased protein accretion rates by 150%. Acute insulin infusion, however, had very little effect on amino acid delivery rates, fetal leucine disposal rates, or fetal protein accretion rates in CON or IUGR fetuses despite robust signaling of the fetal skeletal muscle insulin-signaling cascade. These results indicate that, when amino acids are given directly into the fetal circulation independently of changes in insulin concentrations, IUGR fetal sheep have suppressed protein breakdown rates, thus increasing net fetal protein accretion.     

Effects of methyl-deficient diets on methionine and homocysteine metabolism in the pregnant rat

Although the importance of methyl metabolism in fetal development is well recognized, there is limited information on the dynamics of methionine flow through maternal and fetal tissues and on how this is related to circulating total homocysteine concentrations. Rates of homocysteine remethylation in maternal and fetal tissues on days 11, 19, and 21 of gestation were measured in pregnant rats fed diets with limiting or surplus amounts of folic acid and choline at two levels of methionine and then infused with L-[1-(13)C,(2)H(3)-methyl]methionine. The rate of homocysteine remethylation was highest in maternal liver and declined as gestation progressed. Diets deficient in folic acid and choline reduced the production of methionine from homocysteine in maternal liver only in the animals fed a methionine-limited diet. Throughout gestation, the pancreas exported homocysteine for methylation within other tissues. Little or no methionine cycle activity was detected in the placenta at days 19 and 21 of gestation, but, during this period, fetal tissues, especially the liver, synthesized methionine from homocysteine. Greater enrichment of homocysteine in maternal plasma than placenta, even in animals fed the most-deficient diets, shows that the placenta did not contribute homocysteine to maternal plasma. Methionine synthesis from homocysteine in fetal tissues was maintained or increased when the dams were fed folate- and choline-deficient methionine-restricted diets. This study shows that methyl-deficient diets decrease the remethylation of homocysteine within maternal tissues but that these rates are protected to some extent within fetal tissues.     

Growth and in-vitro metabolism of placental tissues of cows from day 100 to day 250 of gestation.

Weight of placental tissues of cows increased exponentially from Day 100 to Day 250 of gestation, but at much slower relative and absolute rates than fetal weight. In addition, growth rate of fetal placental tissues was less than that of maternal placental tissues. Concentrations of DNA, RNA and protein, however, increased in fetal placental but not in maternal placental tissues. Fetal placental tissues therefore exhibited hyperplasia, which probably contributes to increased functional capacity of the placenta during late gestation. The rate of O2 uptake in vitro was greatest for maternal placental tissues, suggesting that the maternal portion of the placenta accounts for most of the large rate of placental O2 utilization in vivo. Compared with other placental tissues, rate of secretion of macromolecules by intercaruncular endometrium was high, but decreased from Day 100 to 250, suggesting that uterine glandular secretory activity may decrease as gestation advances. Rate of secretion of macromolecules also was high for intercotyledonary tissues and increased with day of gestation, suggesting a role for secretory products of chorioallantois in gravid uterine function.     

Some aspects of amino acid metabolism in the rat fetus

• 1.1. In spite of an eventual catabolic phase during the last third of pregnancy, nitrogen retention seems to increase in pregnant rats. Furthermore, the high uterine blood flow and the high placental transfer of amino acids maintains an adequate nutrient supply to the fetuses.
• 2.2. The terminal rat fetus has a high circulating plasma amino acid level, as well as an increased free amino acid tissue pool when compared to its mother's.
• 3.3. In the rat fetus the development of enzymatic capabilities shows a sudden emergence (also denomined clustering) in late fetal life. In a general trend, the activities of enzymes related with amino acid metabolism are not well developed during rat fetal life.
• 4.4. The rate of amino nitrogen excertion in rat fetus is low, mainly due to the low development of urea cycle enzyme activities.
• 5.5. The rates of protein synthesis in many tissues are high in the rat fetus and they show a progressive decrease until delivery. On the other hand, the rates of protein breakdown are also higher during fetal life than in the adult.

     

Critical Assessment of Methods Used to Measure Protein Synthesis In Human Subjects

The loss of body protein that frequently accompanies illness occurs through changes in protein synthesis and degradation. In human tissues, rates of protein synthesis can be assessed with stable isotopic tracer techniques and mass spectrometry. The basic principles of these methods are explained, and the advantages and drawbacks of the two main approaches, the constant infusion method and the flooding method, are described. Examples are given of the use of these methods to investigate protein synthesis and surgical trauma, pathologies of the gastrointestinal tract and the response of tumor growth to amino acid supplements.     

Carbohydrate Metabolism and Translocation in Healthy and Cocoa Swollen Shoot Virus-Infected Cocoa Plants

Analyses of cocoa swollen shoot virus-infected and healthy cocoa (Theobroma cacao L.) plant tissues were made to determine the effect of virus infection on the metabolism and transport of carbohydrates in affected plants. Starch, sucrose and reducing sugars were found to accumulate in infected tissues. Translocation of photosynthates (mainly as sucrose) to the stem and root system, as estimated by the overnight loss of carbohydrates from the leaves and by ¹⁴CO₂ tracer experiments, was as efficient in the infected plants as in the healthy. Infected plants showed a higher diurnal turnover of carbohydrates in their leaves and, on unit leaf area basis, higher levels of ¹⁴C-labelled assimilates suggesting that they have a greater photosynthetic capacity than the healthy plants. The rate of respiration, as determined by the proportions of organic acids, amino acids and other intermediary metabolites formed from translocated ¹⁴C-labelled sugars, was generally higher in infected than in healthy plants. It is concluded from available data showing the presence in infected tissues of mineral nutrients, protein N and amino acids at the same concentrations as in healthy plants, and from the relatively high rates of photosynthesis and respiration that a high rate of metabolic activity is maintained in the host-virus system. Some factors possibly contributing to the stunted growth of infected plants are discussed in the light of these findings.     

Metabolomic evaluation of di-n-butyl phthalate-induced teratogenesis in mice

Di-n-butyl phthalate (DBP) has been linked to the neural, reproductive and developmental toxicity. We present here a metabolomic study that characterized the metabolic variations associated with the DBP-induced teratogenesis in maternal and fetal mice. DBP at 50 and 300?mg/kg were administrated to pregnant C57 mice, via gastric intubation on gestation day 7?C9, respectively. Maternal mice were euthanized on gestation day 16 and examined for fetal development and malformations. Metabolomic study of maternal serum, placenta and fetal brain tissues was performed using gas chromatography time-of-flight mass spectrometry combined with multivariate data analysis (MVDA). The results showed that a 50?mg/kg dose of DBP had no significant effect on fetal development and a 300?mg/kg dose caused embryo resorption and fetal malformations (primarily eye abnormalities and encephalocele). MVDA indicated that DBP at two doses gave rise to disruption of maternal and fetal metabolic profiles characterized by significantly altered tricarboxylic acid cycle, amino acid, purine and lipid metabolism.     

Effect of maternal hypercholesterolemia on fetal sterol metabolism in the Golden Syrian hamster.

The fetus obtains a significant amount of cholesterol from de novo synthesis. Studies have suggested that maternal cholesterol may also contribute to the cholesterol accrued in the fetus. Thus, the present studies were completed to determine whether diet-induced maternal hypercholesterolemia would affect fetal sterol metabolism. To accomplish this, maternal plasma cholesterol concentrations were increased sequentially by feeding hamsters 0.0%, 0.12%, 0.5%, and 2.0% cholesterol. At 11 days into a gestational period of 15.5 days, cholesterol concentrations and sterol synthesis rates were measured in the three fetal tissues: the placenta, yolk sac, and fetus. In the placenta and yolk sac, the cholesterol concentration increased significantly when dams were fed as little as 0.12% cholesterol (P < 0.0167), and sterol synthesis rates decreased in dams fed at least 0.5% or 2% cholesterol, respectively (P < 0.0167). In the fetus, changes in fetal cholesterol concentration and sterol synthesis rates occurred only when dams were fed at least 0.5% cholesterol, which corresponded to a greater than 2-fold increase in maternal plasma cholesterol concentrations. When the cholesterol concentration in the fetal tissues in each animal was plotted as a function of maternal plasma cholesterol concentration, a linear relationship was found (P < 0.001).These studies demonstrate that sterol homeostasis in fetal tissues, including the fetus, is affected by maternal plasma cholesterol concentration in a gradient fashion and that sterol metabolism in the fetus is dependent on sterol homeostasis in the yolk sac and/or placenta.     

Longitudinal Metabolomic Profiling of Amino Acids and Lipids across Healthy Pregnancy

Pregnancy is characterized by a complexity of metabolic processes that may impact fetal development and ultimately, infant health outcomes. However, our understanding of whole body maternal and fetal metabolism during this critical life stage remains incomplete. The objective of this study is to utilize metabolomics to profile longitudinal patterns of fasting maternal metabolites among a cohort of non-diabetic, healthy pregnant women in order to advance our understanding of changes in protein and lipid concentrations across gestation, the biochemical pathways by which they are metabolized and to describe variation in maternal metabolites between ethnic groups. Among 160 pregnant women, amino acids, tricarboxylic acid (TCA) cycle intermediates, keto-bodies and non-esterified fatty acids were detected by liquid chromatography coupled with mass spectrometry, while polar lipids were detected through flow-injected mass spectrometry. The maternal plasma concentration of several essential and non-essential amino acids, long-chain polyunsaturated fatty acids, free carnitine, acetylcarnitine, phosphatidylcholines and sphingomyelins significantly decreased across pregnancy. Concentrations of several TCA intermediates increase as pregnancy progresses, as well as the keto-body β-hydroxybutyrate. Ratios of specific acylcarnitines used as indicators of metabolic pathways suggest a decreased beta-oxidation rate and increased carnitine palmitoyltransferase-1 enzyme activity with advancing gestation. Decreasing amino acid concentrations likely reflects placental uptake and tissue biosynthesis. The absence of any increase in plasma non-esterified fatty acids is unexpected in the catabolic phase of later pregnancy and may reflect enhanced placental fatty acid uptake and utilization for fetal tissue growth. While it appears that energy production through the TCA cycle increases as pregnancy progresses, decreasing patterns of free carnitine and acetylcarnitine as well as increased carnitine palmitoyltransferase-1 rate and β-hydroxybutyrate levels suggest a concomitant upregulation of ketogenesis to ensure sufficient energy supply in the fasting state. Several differences in metabolomic profiles between Hispanic and non-Hispanic women demonstrate phenotypic variations in prenatal metabolism which should be considered in future studies.     

Insulin-induced alterations in amino acid metabolism in the fetal lamb

To investigate the role of insulin in modulation of fetal amino acid metabolism, insulin infusions were performed in 10 chronically-catheterized fetal lambs. Fetal insulin infusion caused a dose related fall in the arterial blood concentrations of 13 of 15 amino acids studied as well as a 15-25% decrease in total amino acid concentration. Fetal lambs exhibited a biphasic response of umbilical total amino acid uptake when compared to fetal blood insulin concentration, i.e., at achieved fetal insulin concentrations less than 100 microU/ml, umbilical uptake of 9 specific amino acids as well as summed amino acid uptake from the umbilical circulation were depressed, but at insulin concentrations of 100-350 microU/ml, amino acid uptakes were similar to or above control values. Insulin infusion also caused a drastic diminution in the rate of fetal urea excretion. These findings suggest that insulin acts in the fetus to depress amino acid catabolism, thus altering amino acid extraction and uptake. Depressed protein catabolism with or without enhanced amino acid uptake would have the theoretical effect of stimulation of net protein synthesis with a shift toward use of nonprotein substrates for energy purposes.     

Placental permeability and energy metabolism enzymes in fetuses of lipemic rats

A model of maternal lipemia without hyperglycemia, in the rat, produced by high-fat feedings, was developed to study the effects of an abnormal maternal lipid homeostasis on placental transport of nutrients and possible alterations of key enzymes of energy metabolism in the liver and brain of the fetuses. Pregnant rats fed lower concentrations of fat served as controls. All studies were carried out in dams and fetuses one day prior to delivery. The dietary treatment of the dams and fetuses produced in the fetuses ketonemia as well as lipemia. Following a bolus of ¹⁴C-3-0-methyl-D-glucose to the dams, the levels of the tracer remained higher in the blood and brain of lipemic than in control fetuses. By contrast, there was a decrease in the fluxes of ¹⁴C--amino-isobutryic acid in the fetuses of lipemic dams as compared to controls. Among enzymes of energy metabolism, fetal liver glucose-6-phosphatase and succinic dehydrogenase were enhanced by lipemia. Fetal brain glucose-6-phosphatase was depressed. Thus, lipemia, as occuring in poorly controlled maternal diabetes, may be a factor in determining the access to the fetus of essential, neutral amino acids and alter the normal activity of energy metabolism enzymes in the fetus.     

Metabolic flux analysis in mammalian cell culture

Mammalian cell culture metabolism is characterized by glucoglutaminolysis, that is, high glucose and glutamine uptake combined with a high rate of lactate and non-essential amino acid secretion. Stress associated with acid neutralization and ammonia accumulation necessitates complex feeding schemes and limits cell densities achieved in fed-batch culture. Conventional and constraint-based metabolic flux analysis has been successfully used to study the metabolic phenotype of mammalian cells in culture, while ¹³C tracer analysis has been used to study small network models and validate assumptions of metabolism. Large-scale ¹³C metabolic flux analysis, which is required to improve confidence in the network models and their predictions, remains a major challenge. Advances in both modeling and analytical techniques are bringing this challenge within sight.     

Production of slow alpha2-globulin in the pregnant rat.

Slow alpha2-globulin (salpha2G), a high molecular weight glycoprotein, appears as a component of rat serum in a wide variety of both physiologic and pathologic conditions, including pregnancy; it is also present in the neonate. In the present study, the protein was detectable in serum after 6 to 10 days, and was present at moderate levels on day 13, and thereafter through the rest of pregnancy. From immunofluorescent localization and 14C-labelled amino acid incorporation studies, salpha2G was found to be localized in uterine and placental tissues and to be synthesized by these tissues as early as day 6 of gestation. Production continued throughout pregnancy. Synthesis in the liver of the pregnant rat began at day 17 of gestation, which is in contrast with the observation in pathologic conditions that liver synthesis is an initial source of the serum protein; substantial fetal liver synthesis was occurring at day 21 of gestation, and the amount in fetal serum was four times greater than that in maternal serum at that time. It is likely that the fetal liver produces salpha2G as soon as it begins to function.     

Adrenal glands are essential for activation of glucogenesis during undernutrition in fetal sheep near term

In adults, the adrenal glands are essential for the metabolic response to stress, but little is known about their role in fetal metabolism. This study examined the effects of adrenalectomizing fetal sheep on glucose and oxygen metabolism in utero in fed conditions and after maternal fasting for 48 h near term. Fetal adrenalectomy (AX) had little effect on the rates of glucose and oxygen metabolism by the fetus or uteroplacental tissues in fed conditions. Endogenous glucose production was negligible in both AX and intact, sham-operated fetuses in fed conditions. Maternal fasting reduced fetal glucose levels and umbilical glucose uptake in both groups of fetuses to a similar extent but activated glucose production only in the intact fetuses. The lack of fasting-induced glucogenesis in AX fetuses was accompanied by falls in fetal glucose utilization and oxygen consumption not seen in intact controls. The circulating concentrations of cortisol and total catecholamines, and the hepatic glycogen content and activities of key gluconeogenic enzymes, were also less in AX than intact fetuses in fasted animals. Insulin concentrations were also lower in AX than intact fetuses in both nutritional states. Maternal glucose utilization and its distribution between the fetal, uteroplacental, and nonuterine maternal tissues were unaffected by fetal AX in both nutritional states. Ovine fetal adrenal glands, therefore, have little effect on basal rates of fetal glucose and oxygen metabolism but are essential for activating fetal glucogenesis in response to maternal fasting. They may also be involved in regulating insulin sensitivity in utero.     

Kinetic analysis of amino acid pools and protein synthesis in amphibian oocytes and embryos.

Rates of protein synthesis have been measured in Rana pipiens oocytes and embryos and in Xenopus oocytes from the incorporation kinetics of two different concentrations of amino acid. This method does not require an independent measurement of the amino acid pools, since the pool size can be calculated directly from incorporation data. The effects of the concentration and diffusion of injected amino acid on the calculated values for amino acid pool size and flow rate are discussed. When the endogenous amino acid pool is appreciably expanded by the injected amino acid, the total amino acid pool in the oocytes or embryos may be considered as the precursor pool for protein synthesis. Under these circumstances, compartmentation of amino acids does not affect the results, except when lysine is used as tracer. The rates of protein synthesis in ovarian oocytes of Rana pipiens and Xenopus laevis are 18 and 50–54 ng/hr, respectively. In Rana pipiens, the rate increases 70% during maturation and another 50% before the two-cell stage. Finally, the rate approximately doubles between the two-cell and blastula stages.     

Use of stable isotopes to assess protein and amino acid metabolism in children and adolescents: a brief review

As protein accretion is a prerequisite for growth, studying the mechanisms by which nutrients and hormones promote protein gain is of the utmost relevance to paediatric endocrinology. Tracers are ideally suited for the assessment of protein and amino acid kinetics in vivo, as they provide an estimate of synthesis and turnover. Current tracer approaches in children and adolescents utilize stable isotopes, 'heavier' forms of elements that have one or several extra neutrons in the nucleus. Such isotopes are already present at low, but significant, levels in all tissues and foodstuffs, are not radioactive and are devoid of any known side-effects when present in small amounts. L-[1-(13)C] labelled leucine, given as a 4- to 6-h intravenous infusion, has become the method of choice to assess whole-body protein kinetics. After infusion, any 13C-leucine that is oxidized appears in the breath as 13CO2, whereas the remainder is incorporated into body proteins through protein synthesis. The isotope enrichments are determined by isotope ratio mass spectrometry and gas chromatography mass spectrometry, and absolute rates of whole-body protein synthesis, oxidation, and breakdown can be extrapolated. This approach has been used extensively to investigate the regulation of protein kinetics by nutrients and by hormones. Attempts have also been made to measure amino acid/protein metabolism in selected body compartments, and to measure the kinetics of specific tissue proteins, for example, muscle, gut, or plasma proteins.     

The rat visceral yolk sac internalizes maternal transferrin and secretes hydrolyzed products towards the fetus

The uptake of transferrin by the rat visceral yolk sac membranes, and the fate of this protein, were measured in a two-chambered system which allowed access to both surfaces of these membranes, i.e. that facing the maternal compartment and that facing the fetal compartment. 125I-labeled transferrin was internalized by the maternal surface of the visceral yolk sac but not by the fetal surface. Following internalization, this transferrin was degraded and the amino acids were secreted exclusively towards the fetal compartment. Transcytosis of intact transferrin was not detected in either direction. These results suggest that transport across the rat visceral yolk sac bound to maternally derived transferrin is not a major mechanism of iron transport in vivo. These results support a role for the visceral yolk sac in fetal metabolism, or supplying the fetus with amino acids derived from degradation of specific maternal plasma proteins, in this case, transferrin.     

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

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.     

In vivo and in vitro hormonal effects on the metabolism of immature oocytes of Xenopus laevis.

The rate of in vitro amino acid uptake by Xenopus laevis ovarian follicles from hormonally (HCG) stimulated females was compared to that of ovarian follicles from nonstimulated females. An increased rate of uptake was found in HCG-stimulated ovarian follicles. Evidence is presented that indicates that oocytes from HCG-stimulated females have higher protein synthetic rates relative to oocytes from nonstimulated females. When ovarian follicles from unstimulated females were treated with HCG in vitro, it was found that the response obtained mimics the in vivo stimulation both in terms of its effect on amino acid uptake by the ovarian follicles and on the metabolism of the oocyte itself as indicated by increased protein synthetic rates and changes in ribosome metabolism. In order to demonstrate these HCG-mediated changes in oocyte metabolism in vitro, the presence of the entire ovarian follicle was required.