This review presents an overview and recent progress of strategies for detecting isomerism in peptides, with focus on d/l epimerization and the various isomers that the presence of an aspartic acid ...residue may yield in a protein or peptide. While mass spectrometry has become a majorly used method of choice within proteomics, isomerism is inherently difficult to analyze because it is a modification that does not yield any change in mass of the analyte. Here, several techniques used for analysis of peptide isomerism are discussed, including enzymatic assays, liquid chromatography, and capillary electrophoresis. Recent progress in method development using mass spectrometry is also discussed, including labeling strategies, fragmentation techniques, and ion‐mobility spectrometry.
Phthalates are known endocrine disruptors and associated with decreased fecundity, pregnancy loss, and adverse obstetrical outcomes, however the underlying mechanisms remain to be established. ...Environmental factors can influence gene expression and cell function by modifying epigenetic marks, impacting the developing embryo as well as future generations of offspring. The impact of phthalates on placental gene methylation and expression is largely unknown. We studied the effect of maternal phthalate exposure on the human placental DNA methylome and transcriptome. We determined epigenome-wide DNA methylation marks (Illumina Infinium Human Methylation 850k BeadChip) and gene expression (Agilent whole human genome array) associated with phthalate exposure in first trimester placenta. Integrative genomic analysis of candidate genes was performed to define gene methylation-expression relationships. We identified 39 genes with significantly altered methylation and gene expression in the high phthalate exposure group. Most of these relationships were inversely correlated. This analysis identified epidermal growth factor receptor (EGFR) as a critical candidate gene mediating the effects of phthalates on early placental function. Although additional studies are needed to determine the functional consequences of these changes, our findings are consistent with the model that phthalates impact placental function by modulating the expression of critical placental genes through epigenetic regulation.
Intrauterine growth restriction is associated with a range of alterations in placental transport functions: the activity of a number of transporters is reduced (Systems A, L and Tau, transporters for ...cationic amino acids, the sodium-proton exchanger and the sodium pump), placental glucose transporter activity and expression are unchanged whereas the activity of the calcium pump is increased. In contrast, accelerated fetal growth in association to diabetes is characterized by increased activity of placental Systems A and L and glucose transporters. Evidence suggests that these placental transport alterations are the result of specific regulation and that they, at least in part, contribute to the development of pathological fetal growth rather than representing a consequence to altered fetal growth. One interpretation of this data is that the placenta functions as a nutrient sensor, altering placental transport functions according to the ability of the maternal supply line to provide nutrients. Placental transporters are subjected to regulation by hormones. Insulin up-regulates several key placental transporters and maternal insulin may represent a “good nutrition” signal to increase placental nutrient transfer and the growth of the fetus. Preliminary evidence suggests that placental mammalian target of rapamycin, a protein kinase regulating protein translation and transcription in response to nutrient stimuli, may be involved in placental nutrient sensing.
Abstract Adiponectin has well-established insulin-sensitizing effects in non-pregnant individuals. Pregnant women who are obese or have gestational diabetes typically have low circulating levels of ...adiponectin, which is associated with increased fetal growth. Lean women, on the other hand, have high circulating levels of adiponectin. As a result, maternal serum adiponectin is inversely correlated to fetal growth across the full range of birth weights, suggesting that maternal adiponectin may limit fetal growth. In the mother, adiponectin is predicted to promote insulin sensitivity and stimulate glucose uptake in maternal skeletal muscle thereby reducing nutrient availability for placental transfer. Adiponectin prevents insulin-stimulated amino acid uptake in cultured primary human trophoblast cells by modulating insulin receptor substrate phosphorylation. Furthermore, chronic administration of adiponectin to pregnant mice inhibits placental insulin and mammalian target of rapamycin complex 1 (mTORC1) signaling, down-regulates the activity and expression of key placental nutrient transporters and decreases fetal growth. Preliminary findings indicate that adiponectin binds to the adiponectin receptor-2 on the trophoblast cell and activates p38 MAPK and PPAR-α, which inhibits the insulin/IGF-1 signaling pathway. In contrast to maternal adiponectin, recent reports suggest that fetal adiponectin may promote expansion of adipose tissue and stimulate fetal growth. Regulation of placental function by adiponectin constitutes a novel physiological mechanism by which the endocrine functions of maternal adipose tissue influence fetal growth. These findings may help us better understand the factors determining birth weight in normal pregnancies and in pregnancy complications associated with altered maternal adiponectin levels such as obesity and gestational diabetes.
Department of Obstetrics and Gynecology, College of Medicine, University of Cincinnati, Cincinnati, Ohio
Submitted 4 May 2009
; accepted in final form 22 June 2009
Changes in placental nutrient ...transport are closely associated with abnormal fetal growth. However, the molecular mechanisms underlying the regulation of placental amino acid transporters are unknown. We demonstrate that physiological concentrations of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)- stimulate the activity of amino acid transporter system A, but not system L, in cultured human primary trophoblast cells. Both cytokines increased the gene and protein expression of the Na + -coupled neutral amino acid transporter (SNAT)2 isoform and upregulated SNAT1 protein expression. IL-6 increased Tyr705 phosphorylation of signal transducer and activator of transcription 3 (STAT3). In cells transfected with small interfering RNA (siRNA) targeting STAT3, the RNA and protein expression of SNAT2, but not SNAT1, was reduced and the stimulating effect of IL-6 on system A activity was abolished. Despite eliciting similar responses in amino acid transport activity and transporter expression, TNF- effects on system A activity were not mediated through the JAK/STAT pathway. In conclusion, we have identified a novel regulatory pathway involving increased gene expression of the SNAT2 isoform mediated by a STAT-dependent pathway, which links IL-6 to increased activity of system A, a ubiquitously expressed transporter of neutral amino acids. From these new findings, we propose that upregulation of amino acid transporters by cytokines may contribute to increased placental nutrient transport and fetal overgrowth, which are commonly found in pregnancies complicated by maternal diabetes and obesity.
placenta; pregnancy; cytokines; obesity
Address for reprint requests and other correspondence: H. N. Jones, The Center for Molecular Fetal Therapy, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229 (e-mail: helen.jones{at}cchmc.org ).
Intrauterine growth restriction (IUGR) represents an important risk factor for perinatal complications and for adult disease.
IUGR is associated with a down-regulation of placental amino acid ...transporters; however, whether these changes are primary
events directly contributing to IUGR or a secondary consequence is unknown. We investigated the time course of changes in
placental and fetal growth, placental nutrient transport in vivo and the expression of placental nutrient transporters in pregnant rats subjected to protein malnutrition, a model for IUGR.
Pregnant rats were given either a low protein (LP) diet ( n = 64) or an isocaloric control diet ( n = 66) throughout pregnancy. Maternal insulin, leptin and IGF-I levels decreased, whereas maternal amino acid concentrations
increased moderately in response to the LP diet. Fetal and placental weights in the LP group were unaltered compared to control
diet at gestational day (GD) 15, 18 and 19 but significantly reduced at GD 21. Placental system A transport activity was reduced
at GD 19 and 21 in response to a low protein diet. Placental protein expression of SNAT2 was decreased at GD 21. In conclusion,
placental amino acid transport is down-regulated prior to the development of IUGR, suggesting that these placental transport
changes are a cause, rather than a consequence, of IUGR. Reduced maternal levels of insulin, leptin and IGF-1 may link maternal
protein malnutrition to reduced fetal growth by down-regulation of key placental amino acid transporters.
Cell-to-cell variability and functional heterogeneity are integral features of multicellular organisms. Chemical classification of cells into cell type is important for understanding cellular ...specialization as well as organismal function and organization. Assays to elucidate these chemical variations are best performed with single cell samples because tissue homogenates average the biochemical composition of many different cells and oftentimes include extracellular components. Several single cell microanalysis techniques have been developed but tend to be low throughput or require preselection of molecular probes that limit the information obtained. Mass spectrometry (MS) is an untargeted, multiplexed, and sensitive analytical method that is well-suited for studying chemically complex individual cells that have low analyte content. In this work, populations of cells from the rat pituitary, the rat pancreatic islets of Langerhans, and from the Aplysia californica nervous system, are classified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI) MS by their peptide content. Cells were dispersed onto a microscope slide to generate a sample where hundreds to thousands of cells were separately located. Optical imaging was used to determine the cell coordinates on the slide, and these locations were used to automate the MS measurements to targeted cells. Principal component analysis was used to classify cellular subpopulations. The method was modified to focus on the signals described by the lower principal components to explore rare cells having a unique peptide content. This approach efficiently uncovers and classifies cellular subtypes as well as discovers rare cells from large cellular populations.
Apelin is an insulin-sensitizing hormone increased in abundance with obesity. Apelin and its receptor, APJ, are expressed in the human placenta, but whether apelin regulates placental function in ...normal body mass index (BMI) and obese pregnant women remains unknown. We hypothesized that apelin stimulates amino acid transport in cultured primary human trophoblast (PHT) cells and that maternal circulating apelin levels are elevated in obese pregnant women delivering large babies. Treating PHT cells with physiological concentrations of the pyroglutamated form Pyr
apelin-13 (0.1-10.0 ng/ml) for 24 h dose-dependently increased System A amino acid transport (
< 0.05) but did not affect System L transport activity. Mechanistic target of rapamycin (mTOR), extracellular signal-regulated kinase-1/2 (ERK1/2), and AMP-activated protein kinase-α (AMPKα) signaling were unaffected by apelin (
> 0.05). Plasma apelin was not different in obese women (BMI 35.8 ± 0.7,
= 21) with large babies compared with normal-BMI women (23.1 ± 0.5,
= 16) delivering normal birth weight infants. Apelin was highly expressed in placental villous tissue (20-fold higher vs. adipose), and APJ was present in syncytiotrophoblast microvillous membrane, but neither differed in abundance between normal-BMI and obese women. Phosphorylation (Thr
) of placental AMPKα strongly correlated with microvillous membrane APJ expression (
< 0.01,
= 0.63) but negatively correlated with placental apelin abundance (
< 0.01,
= -0.62). Neither placental APJ nor apelin abundance correlated with maternal BMI, plasma insulin, birth weight, or mTOR or ERK1/2 signaling (
> 0.05). Hence, apelin stimulates trophoblast amino acid uptake, establishing a novel mechanism regulating placental function. We found no evidence that apelin constitutes an endocrine link between maternal obesity and fetal overgrowth.
1 Perinatal Center, Department of Physiology, Institute of Neuroscience and Physiology, University of Gothenburg, Gothenburg, Sweden;
2 Department of Obstetrics and Gynecology, Institute of Clinical ...Sciences, University of Gothenburg, Gothenburg, Sweden; and
3 Department of Obstetrics and Gynecology, University of Cincinnati College of Medicine, Cincinnati, Ohio
Submitted 30 April 2009
; accepted in final form 3 July 2009
Inhibition of mammalian target of rapamycin (mTOR) signaling in cultured human primary trophoblast cells reduces the activity of key placental amino acid transporters. However, the upstream regulators of placental mTOR are unknown. We hypothesized that glucose, insulin, and IGF-I regulate placental amino acid transporters by inducing changes in mTOR signaling. Primary human trophoblast cells were cultured for 24 h with media containing various glucose concentrations, insulin, or IGF-I, with or without the mTOR inhibitor rapamycin, and, subsequently, the activity of system A, system L, and taurine (TAUT) transporters was measured. Glucose deprivation (0.5 mM glucose) did not significantly affect Thr172-AMP-activated protein kinase phosphorylation or REDD1 expression but decreased S6 kinase 1 phosphorylation at Thr389. The activity of system L decreased in a dose-dependent manner in response to decreasing glucose concentrations. This effect was abolished in the presence of rapamycin. Glucose deprivation had two opposing effects on system A activity: 1 ) an "adaptive" upregulation mediated by an mTOR-independent mechanism and 2 ) downregulation by an mTOR-dependent mechanism. TAUT activity was increased after incubating cells with glucose-deprived media, and this effect was largely independent of mTOR signaling. Insulin and IGF-I increased system A activity and insulin stimulated system L activity, effects that were abolished by rapamycin. We conclude that the mTOR pathway represents an important intracellular regulatory link between nutrient and growth factor concentrations and amino acid transport in the human placenta.
placental transport; insulin; insulin-like growth factor I; mammalian target of rapamycin
Address for reprint requests and other correspondence: S. Roos, Perinatal Center, Dept. of Physiology, Institute of Neuroscience and Physiology, Univ. of Gothenburg, P.O. Box 432, SE-405 30 Gothenburg, Sweden (e-mail: sara.roos{at}gu.se ).
A receptor binding class of d-amino acid-containing peptides (DAACPs) is formed in animals from an enzymatically mediated post-translational modification of ribosomally translated all-l-amino acid ...peptides. Although this modification can be required for biological actions, detecting it is challenging because DAACPs have the same mass as their all-l-amino acid counterparts. We developed a suite of mass spectrometry (MS) protocols for the nontargeted discovery of DAACPs and validated their effectiveness using neurons from Aplysia californica. The approach involves the following three steps, with each confirming and refining the hits found in the prior step. The first step is screening for peptides resistant to digestion by aminopeptidase M. The second verifies the presence of a chiral amino acid via acid hydrolysis in deuterium chloride, labeling with Marfey’s reagent, and liquid chromatography–mass spectrometry to determine the chirality of each amino acid. The third involves synthesizing the putative DAACPs and comparing them to the endogenous standards. Advantages of the method, the d-amino acid-containing neuropeptide discovery funnel, are that it is capable of detecting the d-form of any common chiral amino acid, and the first two steps do not require peptide standards. Using these protocols, we report that two peptides from the Aplysia achatin-like neuropeptide precursor exist as GdYFD and SdYADSKDEESNAALSDFA. Interestingly, GdYFD was bioactive in the Aplysia feeding and locomotor circuits but SdYADSKDEESNAALSDFA was not. The discovery funnel provides an effective means to characterize DAACPs in the nervous systems of animals in a nontargeted manner.
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