Ontogenic Regulation Research Articles

Recent genome duplications facilitate the phenotypic diversity of Hb repertoire in the Cyprinidae.

Whole-genome duplications (WGDs) are an important contributor to phenotypic innovations in evolutionary history. The diversity of blood oxygen transport traits is the perfect reflection of physiological versatility for evolutionary success among vertebrates. In this study, the evolutionary changes of hemoglobin (Hb) repertoire driven by the recent genome duplications were detected in representative Cyprinidae fish, including eight diploid and four tetraploid species. Comparative genomic analysis revealed a substantial variation in both membership composition and intragenomic organization of Hb genes in these species. Phylogenetic reconstruction analyses were conducted to characterize the evolutionary history of these genes. Data were integrated with the expression profiles of the genes during ontogeny. Our results indicated that genome duplications facilitated the phenotypic diversity of the Hb gene family; each was associated with species-specific changes in gene content via gene loss and fusion after genome duplications. This led to repeated evolutionary transitions in the ontogenic regulation of Hb gene expression. Our results revealed that genome duplications helped to generate phenotypic changes in Cyprinidae Hb systems.

Developmental expression profiles and thyroidal regulation of cytokines during metamorphosis in the amphibian Xenopus laevis

Early life-stages of amphibians rely on the innate immune system for defense against pathogens. While thyroid hormones (TH) are critical for metamorphosis and later development of the adaptive immune system, the role of TH in innate immune system development is less clear. An integral part of the innate immune response are pro-inflammatory cytokines – effector molecules that allow communication between components of the immune system. The objective of this study was to characterize the expression of key pro-inflammatory cytokines, tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β) and interferon-γ (IFN-γ), throughout amphibian development and determine the impacts of thyroidal modulation on their expression. Xenopus laevis were sampled at various stages of development encompassing early embryogenesis to late prometamorphosis and cytokine expression was measured by real-time PCR. Expression of TNFα and IL-1β were transient over development, increasing with developmental stage, while IFN-γ remained relatively stable. Functionally athyroid, premetamorphic tadpoles were exposed to thyroxine (0.5 and 2 μg/L) or sodium perchlorate (125 and 500 μg/L) for seven days. Tadpoles demonstrated characteristic responses of advanced development with thyroxine exposure and delayed development (although to a lesser extent) and increased thyroid gland area and follicular cell height with sodium perchlorate exposure. Exposure to thyroxine for two days resulted in decreased expression of IL-1β in tadpole trunks. Sodium perchlorate had negligible effects on cytokine expression. Overall, these results demonstrate that cytokine transcript levels vary with stage of tadpole development but that their ontogenic regulation is not likely exclusively influenced by thyroid status. Understanding the direct and indirect effects of altered hormone status may provide insight into potential mechanisms of altered immune function during amphibian development.

A single element in the 3'UTR of the apical sodium-dependent bile acid transporter controls both stabilization and destabilization of mRNA.

mRNA stability appears to play a key role in the ontogenic regulation of the apical sodium-dependent bile acid transporter (ASBT). The RNA-binding proteins Hu antigen R (HuR) and tristetraprolin (TTP) stabilize and destabilize ASBT mRNA, respectively. Potential HuR-binding sites were assessed by sequence analysis in the context of prior invitro functional analyses of the rat ASBT 3'UTR. Wild-type and mutant-binding sites were investigated by gel-shift analysis using IEC-6 cell extracts. The functional consequences of binding site mutations were assessed using two different hybrid reporter constructs linking the 3'UTR element to either a luciferase or a β-globin coding mRNA sequence. A specific metastasis-associated gene 1 (MTA1) cis-element was identified in the ASBT 3'UTR that became associated with proteins in IEC-6 cell extracts and could be supershifted by anti-HuR or anti-TTP antibodies. Mutation of this cis-element abrogated the gel shift of IEC-6 proteins. Furthermore, hybrid constructs containing a mutant MTA1 element had reduced responses to modulation of HuR or TTP. For the first time, we have identified a single specific sequence element in the 3'UTR of the rat ASBT mRNA that mediates counter-regulatory changes in mRNA abundance in response to both HuR and TTP.

Aliskiren toxicity in juvenile rats is determined by ontogenic regulation of intestinal P-glycoprotein expression

Juvenile rat toxicity studies with the direct renin inhibitor aliskiren were initiated to support treatment in the pediatric population. In Study 1, aliskiren was administered orally to juvenile rats at doses of 0, 30, 100 or 300mg/kg/day with repeated dosing from postpartum day (PPD) 8 to PPD 35/36. In-life, clinical pathology, anatomic pathology, and toxicokinetics evaluations were performed. In Study 2, single oral doses of aliskiren (0, 100 or 300mg/kg) were given to 14-, 21-, 24-, 28-, 31- or 36-day-old rats; in-life data and toxicokinetics were evaluated. Study 3 was a single dose (3mg/kg i.v.) pharmacokinetic study in juvenile rats on PPD 8, 14, 21 and 28. In Study 4, naïve rats were used to investigate ontogenic changes of the multidrug-resistant protein 1 (MDR1) and the organic anion transporting polypeptide (OATP) mRNA in several organs. Oral administration of aliskiren at 100 and 300mg/kg caused unexpected mortality and severe morbidity in 8-day-old rats. Aliskiren plasma and tissue concentrations were increased in rats aged 21days and younger. Expression of MDR1 and OATP mRNA in the intestine, liver and brain was significantly lower in very young rats. In conclusion, severe toxicity and increased exposure in very young rats after oral administration of aliskiren are considered to be the result of immature drug transporter systems. Immaturity of MDR1 in enterocytes appears to be the most important mechanism responsible for the high exposure.

Dietary and ontogenic regulation of fatty acid desaturase and elongase expression in broiler chickens

Effects of diet and ontogeny on the expression of fatty acid desaturases and elongases were examined in broiler chickens. In Study 1, 120 day-old male chicks received one of six diets with LA:ALA ranging from 46:4 to 16:34, for 33 days. Total n−6 PUFA decreased, and n−3 PUFA increased in response to a decrease in the dietary LA:ALA. FADS1, FADS2, ELOVL2 and ELOVL5 mRNAs were highest (P<0.05) in birds fed lower LA:ALA diets. In Study 2, 60 day-old male chicks were fed a basal diet, and liver samples were collected on day of hatch, and on days 2, 7, 14, 21 and 35 post-hatch. Total n−6 and n−3 PUFA increased (P<0.01) from days 7 to days 21. FADS1, FADS2 and ELOVL2 mRNAs generally increased (P<0.01) with age. These findings provide evidence for the dietary and developmental regulation of PUFA metabolism in broiler chickens.

Ontogeny of the corticotropin-releasing factor system in rainbow trout and differential effects of hypoxia on the endocrine and cellular stress responses during development

To further our understanding of the development of the stress axis and the responsiveness of embryonic and larval fish to environmental stressors, this study examined the ontogeny of whole-body cortisol levels and of the corticotropin-releasing factor (CRF) system in rainbow trout, as well as the endocrine and cellular stress responses to hypoxia. After depletion of a maternal deposit, de novo synthesis of cortisol increased exponentially between the ‘eyed’ stage and first feeding. Whole body CRF mRNA levels dominated over those of the related peptide urotensin I (UI) from hatch through complete yolk sac absorption. The mRNA levels of CRF-binding protein (CRF-BP) closely paralleled those of CRF and UI throughout ontogeny except at first feeding when an increase in CRF gene expression was not matched by change in CRF-BP transcript abundance. In the hypoxia challenge, fish were exposed to 15% O2 saturation for either 90min or 24h at three key developmental stages: hatch, swim up and first feeding. While the embryos were unaffected, chronic hypoxia elicited a transient 2-fold increase in whole-body cortisol levels in the larval stages. The hypoxia challenge also generally suppressed the mRNA levels of CRF and CRF-BP, had no effect on the expression of UI, but had a marked stimulatory effect on heat shock protein 70 (Hsp70) gene expression. Taken together, these results suggest a role for the CRF system in the ontogenic regulation of corticosteroidogenesis and show that hypoxia has developmental stage-specific effects on the endocrine and cellular stress responses in rainbow trout.

Ontogeny of d-Mannose Transport and Metabolism in Rat Small Intestine

Oral mannose therapy is used to treat congenital disorders of glycosylation caused by a deficiency in phosphomannose isomerase. The segmental distribution and ontogenic regulation of D-mannose transport, phosphomannose isomerase, and phosphomannose mutase is investigated in the small intestine of fetuses, newborn, suckling, 1-month-old, and adult rats. The small intestine transports D-mannose by both Na(+)-dependent and Na(+)-independent transport mechanisms. The activities of both systems normalized to intestinal weight peak at birth and thereafter they decreased. In all the ages tested, the activity of the Na(+)-independent mechanism was higher than that of the Na(+)/mannose transport system. At birth, the Na(+)-independent D-mannose transport in the ileum was significantly higher than that in jejunum. Phosphomannose isomerase activity and mRNA levels increased at 1 month, and the values in the ileum were lower than in jejunum. Phosphomannose mutase activity in jejunum increased during the early stages of life, and it decreased at 1 month old, as does the amount of mannose incorporated into glycoproteins, whereas in the ileum, they were not affected by age. The phosphomannose isomerase/phosphomannose mutase activity ratio decreased at birth and during the suckling period, and increased at 1 month old. In conclusion, intestinal D-mannose transport activity and metabolism were affected by ontogeny and intestinal segment.

Gnotobiotic miniature swine for immunobiology and medicine

The immune system is a body’s defense system, essential for survival of all vertebrates. It defends not only against invading bacterial, viral, richettsial, fungal and parasitic infections and toxins, and any other intrusions from outsiders, but also provides for surveillance against aberrant endogenous cells which are deviates from normal cells within the host (i.e. cancer), as well as clearing damaged and aging cells, and repairing and maintaining the other tissues. It also has a self-monitoring system for its own defense system to maintain its immunological balance. Over the last 40 years we have been investigating the ontogenic development and regulation of the immune response using the gnotobiotic miniature swine model. We found varying degrees of placental leakage of maternal immunoglobulin in the herds of Minnesota miniature swine and, therefore, constructed a barrier-sustained, specific pathogen-free (SPF)/gnotobiotic miniature swine facility with Hepa-filtered air, sterile water, and sterile feed and maintained under positive pressure. Germ-free (GF) colostrum-deprived piglets obtained by aseptic hysterectomy from Minnesota miniature swine 23 days before term were maintained in GF isolators and associated with normal flora (anaerobic Lactobacillus sp. and anaerobic Streptococcus sp.) and transported into the SPF/gnotobiotic miniature swine facility. We established the breeding and maintenance of SPF/ gnotobiotic miniature swine herds with healthy, nonleaky placental barriers. Thus, gnotobiotic miniature swine are an excellent animal model for immunobiology and medicine. We have demonstrated that: (1) GF colostrumdeprived piglets obtained by aseptic hysterectomy from SPF/gnotobiotic miniature swine are immunologically ‘virgin’ as indicated by the absence of

Ontogenic development-associated changes in the expression of genes involved in rat bile acid homeostasis

Ontogenic changes in the rat bile acid (BA) pool, measured enzymatically and by GC-MS, and expression of enzymes (5alpha-reductase, 5beta-reductase, and cytochrome P450 enzymes Cyp7a1, Cyp8b1, Cyp27 and Cyp3a11), transporters [bile salt export pump, sodium taurocholate-cotransporting polypeptide, apical sodium-dependent bile acid transporter, and organic solute transporter alpha/beta (Ostalpha/Ostbeta)], and nuclear receptors [fetoprotein transcription factor (Ftf), farnesoid X receptor (Fxr), small heterodimer partner (Shp), and hepatic nuclear factor 4alpha (HNF-4alpha)], determined by quantitative PCR, were investigated. The absolute size of the BA pool increased progressively up to adulthood, whereas the complexity of its composition was high in fetuses, decreased after birth, increased again progressively up to adulthood, and decreased in aged animals. Allo-cholic acid only appeared early in development, in spite of low 5alpha-reductase expression. The relative size of the BA pool, corrected by liver weight, was maintained from 1 week after birth, except at weaning, when a transient peak accompanied by Shp downregulation and Cyp7a1 upregulation was observed. An imposed weaning delay of 1 week had no effect on the time course of the BA pool size but decreased the proportion of chenodeoxycholic and alpha-muricholic acids, whereas the proportion of cholic acid was increased, probably as a result of Cyp8b1 upregulation. In conclusion, changes in the expression of genes involved in BA homeostasis may play a role in physiological adaptations to digestive functions during the rat life span.

Effects of age and mineral intake on the regulation of iron absorption in infants

Iron deficiency anemia during infancy is associated with delayed psychomotor and cognitive development; thus there is global interest in improving infant iron status through iron supplementation programs early in life. In adults, iron homeostasis primarily is controlled through regulatory changes in iron absorption; however, there is little understanding of mechanisms that regulate iron absorption in infants. As iron stores become depleted from rapid hemoglobin expansion, ontogenic transition to regulated iron absorption must occur. To elucidate age-dependent mechanisms regulating intestinal iron absorption and homeostasis in human infants, studies in suckling rat pups indicated that increased divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1), and hephaestin expression and changes in localization within the enterocyte may direct the ontogenic regulation of iron homeostasis mechanisms. Unlike observations in adults, iron absorption, DMT1, and FPN1 expression were not affected by iron intake during mid-infancy, similar to observations in human infants. Interestingly, at this age supplemental iron was retained in the small intestine, but only in pups that were not previously iron deficient. Although intestinal retention may possibly help limit excess iron absorption, decreased linear growth, head circumference, and weight gain has been observed in iron-supplemented, nonanemic human infants, suggesting that global iron supplementation programs should be approached with caution.

Ontogenic Profiling of Glucosinolates, Flavonoids, and Other Secondary Metabolites in Eruca sativa (Salad Rocket), Diplotaxis erucoides (Wall Rocket), Diplotaxis tenuifolia (Wild Rocket), and Bunias orientalis (Turkish Rocket)

As an influence of the Mediterranean diet, rocket species such as Eruca sativa L., Diplotaxis species, and Bunias orientalis L. are eaten all over the world at different ontogenic stages in salads and soups. They are all species within the plant order Capparales (glucosinolate-containing species), and all are from the family Brassicaceae. Predominantly, the leaves of these species are eaten raw or cooked, although Eruca flowers are also consumed. There is considerable potential with raw plant material for a higher exposure to bioactive phytochemicals such as glucosinolates, their hydrolysis products, and also phenolics, flavonoids, and vitamins such as vitamin C. These compounds are susceptible to ontogenic variation, and the few published studies that have addressed this topic have been inconsistent. Thus, an ontogenic study was performed and all samples were analyzed using a previously developed robust liquid chromatography/mass spectrometry method for the identification and quantification of the major phytochemicals in all tissues of the rocket species. Seeds and roots of both Eruca and Diplotaxis contained predominantly 4-methylthiobutylglucosinolate. Leaves of Eruca and Diplotaxis contained high amounts of 4-mercaptobutylglucosinolate with lower levels of 4-methylthiobutlyglucosinolate and 4-methylsulfinylbutylglucosinolate. Flowers of Eruca and Diplotaxiscontained predominantly 4-methylsulfinylbutyl-glucosinolate. In addition, roots of both Diplotaxisspecies contained 4-hydroxybenzylglucosinolate but 4-hydroxybenzylglucosinolate was absent from roots of Eruca. Seeds and seedlings of all Eruca contained N-heterocyclic compounds but no sinapine, whereas Diplotaxis contained sinapine but not the N-heterocycles. In all tissues of B. orientalis, 4-hydroxybenzylglucosinolate and 4-methylsulfinyl-3-butenylglucosinolate were predominant. All rocket tissues, except roots, contained significant levels of polyglycosylated flavonoids, with/without hydroxycinnamoyl acylation. The core aglycones were kaempferol, quercetin, and isorhamnetin. The exception was B. orientalis, which had a negligible seed flavonoid content as compared with the other species. Anthocyanins were only detected in Eruca flowers and consisted of a complex pattern of at least 16 different anthocyanins.

Intra‐ and interindividual variability of glucuronidation of paracetamol during repeated administration of propacetamol in neonates

Major changes in drug clearance and metabolism are observed during infancy, in part based on ontogenic regulation of various metabolic pathways. Since paracetamol provides a good substrate to study UGT (1A6) activity, urinary metabolites of propacetamol were determined in neonates in whom propacetamol was repeatedly administered. Paracetamol glucuronide (APAP-G), paracetamol sulphate (APAP-S) and free paracetamol were determined in urine samples of neonates during repeated administration of propacetamol. Spearman rank and linear multiple regression (MedCalc, Mariakerke, Belgium) were used to study the effect of postnatal age, of postconceptional age and of repeated administration on the relative contribution of APAP-G to overall urine paracetamol (APAP-G+APAP-S+free paracetamol) elimination (G/T ratio). 147 samples were collected in 23 neonates. Molar median G/T ratio was 14% (range 1-53). Besides increasing G/T ratio with increasing postnatal (p<0.0001) and postconceptional age (p<0.01), repeated administration (p<0.01) also correlated with an increasing G/T ratio, and repeated administration remained significant (p<0.01) after correction of postnatal and postconceptional age in a multiple regression model. Major variability in the ontogeny of UGT activity to overall elimination of paracetamol was documented in neonates. Besides postnatal and postconceptional age, a significant effect of repeated administration on UGT activity was documented.

Multiple mechanisms of ontogenic regulation of nuclear receptors during rat liver development.

Nuclear receptors (NRs) play pivotal roles in the regulation of genes contributing to hepatobiliary cholesterol and bile acid homeostasis. We have previously shown that transporters involved in bile formation are developmentally regulated and are poorly developed during the fetal stage, but their expression reached gradual maturity during the postnatal period. To define the molecular mechanisms underlying this regulation and the role that class II NRs and associated members [liver receptor homolog-1 (LRH-1) and short heterodimer partner (SHP)] play, we have analyzed the ontogeny of NR expression during liver development. Real-time PCR analysis of hepatic NR expression from fetal day 17 through adult revealed that steady-state mRNA levels for all NRs were very low during the embryonic period. However, mRNA levels peaked close to that of adult rats (>6 wk-old rats) by 4 wk of age for farnesoid X receptor (FXR), pregnane X receptor (PXR), liver X receptor-alpha (LXRalpha), peroxisome proliferator-activated receptor-alpha (PPARalpha), retinoid acid receptor-alpha (RARalpha), LRH-1, and SHP, whereas RXRalpha mRNA lagged behind. FXR, PXR, LXRalpha, RARalpha, and PPARalpha functional activity in liver nuclear extracts assayed by gel EMSA demonstrated that the activity attained adult levels by 4 wk of age, exhibiting a strict correlation with mRNA levels. Surprisingly, PPARalpha activity was delayed as seen by EMSA assay. Protein levels for NRs also corresponded to the mRNA and functional activity except for RXRalpha. RXRalpha protein levels were higher than message levels, suggesting increased protein stability. We conclude that expression of NRs during rat liver development is primarily regulated by transcriptional mechanisms, which in turn, control the regulation of bile acid and cholesterol metabolic pathways.

7 Ontogeny of Udp-Glucuronosyltransferase Activity During Repeated Administration of Propacetamol in Neonates

Background: Major changes in drug clearance are observed during infancy, at least partially based on ontogenic regulation of metabolic pathways but data on maturation of UDP-glucuronosyltransferase (UGT)-activity in term and especially in preterm neonates are still limited. Since paracetamol provides a good substrate to study ontogeny of UGT-1A6 activity, urinary metabolites of propacetamol were determined in neonates in whom repeated administration of propacetamol (a prodrug of paracetamol for intravenous use) was part of their routine clinical care.Methods: Following informed parental consent, urine samples were collected during consecutive time intervals in neonates who received consecutive administrations of propacetamol. In these urine samples, paracetamol-glucuronide, paracetamol-sulfate and free paracetamol were determined using High Performance Liquid Chromatography. Spearman rank and linear multiple regression (MedCalc®) were used to study correlations of postnatal age, of postmenstrual age and of repeated administration on the relative contribution of paracetamol-glucuronide to overall urine paracetamol elimination (G/T ratio).Results: 142 samples were collected in 18 neonates with a median postnatal age of 19 days (range 1–173) and a median postmenstrual age of 39 weeks (range 29–60). Median urine G/T ratio was 14 % (range 1–53). Besides increasing G/T ratio with increasing postnatal (p<0.0001) and postmenstrual age (p<0.01), repeated administration (p<0.01) also correlated with an increasing G/T ratio and this observation remained significant (p<0.01) when these 3 perinatal variables were entered in a multiple linear regression model.Conclusions: The range of urine G/T ratio's documented in this study reflects the major variability in the ontogeny of UGT-activity in neonates of various postnatal and postmenstrual ages. Besides the significant effect of postnatal and postmenstrual age on UGT-activity, an additional independent significant effect of repeated administration of propacetamol was documented. Therefore, observed variability in paracetamol clearance can not only be explained by postnatal and postmenstrual age, but also in part by the number of administered doses of this drug, reflecting an inductive effect of paracetamol on UGT-activity.

Ontogenic regulation of folate transport across rat jejunal brush-border membrane.

Folate is an essential micronutrient that in mammals must be obtained from exogenous sources via intestinal absorption. Previous studies from our laboratory and others have demonstrated that folate absorption from the small intestine is mediated via the reduced-folate carrier (RFC). The goal of this study was to determine whether the initial step of folate uptake by intestinal epithelial cells, i.e., transport across the brush-border membrane (BBM) of the polarized enterocytes, is ontogenically regulated, and if so, to determine the molecular mechanism involved. Purified BBM vesicles (BBMV) isolated from suckling, weanling, and adult rats were used in this study. The initial rate of carrier-mediated uptake of a physiological concentration of folic acid (0.1 muM) by jejunal BBMV was found to be significantly (P < 0.01) higher in suckling compared with weanling rats, which was, in turn, significantly (P < 0.01) higher than that in adult rats. This decline in carrier-mediated folate uptake with maturation was found to be mediated via a decrease in the maximum velocity of the folate uptake process (6.55 +/- 0.87, 2.16 +/- 0.10, 0.90 +/- 0.16 pmol.mg protein-1.10 s-1 for suckling, weanling, and adult rats, respectively), with no changes in its apparent Km. Western blot analysis of BBM protein and real-time PCR showed RFC protein and mRNA levels, respectively, to be significantly (P < 0.01 for both) higher in suckling compared with weanling rats, which were in turn significantly (P < 0.01 for both) higher than that in adult rats. These changes were found by nuclear run-on assay to be associated with a parallel decline in the RFC transcriptional rate in jejunal epithelia with maturation. In situ hybridization showed a similar pattern of RFC message distribution along crypt/villus axis in suckling and adult rat jejunum. These results demonstrate for the first time that folate transport across the intestinal BBM is under ontogenic regulation during early stages of life and that this regulation involves a transcriptional regulatory mechanism(s).

Ontogenic regulation of components of ileal bile acid absorption.

The apical sodium-dependent bile acid cotransporter (ASBT) and the ileal bile acid binding protein (IBABP) are two components of ileal bile acid absorption. During the third postnatal week of the rat, there is a dramatic increase in ASBT and IBABP expression. The goals of this study were to examine the role of hormones on the ontogenic expression of ASBT mRNA and the role of weaning for both ASBT and IBABP mRNA. Administration of various doses of dexamethasone during the second postnatal week induced ASBT mRNA levels, and this effect was significantly increased with concomitant thyroxine treatment. Early weaning and weaning prevention were utilized to investigate the influence of dietary factors. ASBT and IBABP mRNA levels were significantly elevated by early weaning and were decreased by weaning prevention compared with littermate controls. Thus, glucocorticoids and thyroxine appear to play a role in the ontogenic expression of ASBT mRNA and weaning appears to participate in both ASBT and IBABP expression.

Ontogeny of the glucagon-like peptide-2 receptor axis in the developing rat intestine.

Glucagon-like peptide-2 (GLP-2) is secreted by enteroendocrine cells in the small and large intestines and exerts intestinotropic effects in the gastrointestinal mucosal epithelium of the adult rodent. The actions of GLP-2 are mediated by the GLP-2 receptor, a new member of the G protein-coupled receptor superfamily. To ascertain whether the GLP-2/GLP-2 receptor axis is expressed and functional in the developing intestine, we have studied the synthesis of GLP-2 and the expression of the GLP-2 receptor (GLP-2R) in the fetal and neonatal rat gut. GLP-2 immunoreactivity (GLP-2-IR) was detected in the fetal rat intestine, and fetal rat intestinal cell cultures secreted correctly processed GLP-2(1-33) into the medium. High levels of GLP-2(1-33) were also detected in the circulation of 13-day-old neonatal rats (P < 0.001 vs. adult). Analysis of GLP-2 receptor expression by RT-PCR demonstrated GLP-2R messenger RNA transcripts in fetal intestine and in neonatal stomach, jejunum, ileum, and colon. The levels of GLP-2R messenger RNA transcripts were comparatively higher in the fetal and neonatal intestine (P < 0.05-001 vs. adult) and declined to adult levels by postnatal day 21. Subcutaneous administration of a degradation-resistant GLP-2 analog, h[Gly2]-GLP-2 once daily for 10 days increased stomach (0.009 +/- 0.0003 vs. 0.007 +/- 0.002 g/g body mass, h[Gly2]-GLP-2-treated vs. controls; P < 0.05) and small bowel weight (0.043 +/- 0.0037 vs. 0.031 +/- 0.0030 g/g body mass; P < 0.05). h[Gly2]-GLP-2 also increased both small (2.4 +/- 0.05 vs. 1.8 +/- 0.17 cm/g body mass; P < 0.05) and large bowel length (0.32 +/- 0.01 vs. 0.25 +/- 0.02 cm/g body mass, h[Gly2]-GLP-2-treated vs. saline-treated controls, respectively; P < 0.05) in neonatal rats. These findings demonstrate that both components of the GLP-2/GLP-2 receptor axis are expressed in the fetal and neonatal intestine. The ontogenic regulation and functional integrity of this axis raises the possibility that GLP-2 may play a role in the development and/or maturation of the developing rat intestine.

Glucuronidation in Humans

During human development impressive changes in drug disposition occur. An important determinant of drug clearance is metabolism, something that is not only determined by ontogenic regulation but also by genetic processes which add to the variability of drug metabolism during different stages of childhood. Therefore, an understanding of the developmental regulation of different metabolic pathways, together with information on the genetic determinants of drug metabolism, will increase the knowledge of inter- and intraindividual variability in drug disposition during childhood. Conjugation has historically received less attention than cytochrome P450 metabolism. An important group of conjugation reactions are catalysed by the uridine 5'-diphosphate (UDP)-glucuronosyltransferases (UGTs); to date at least 10 different UGT isoforms have been identified. The UGTs are not only involved in the metabolism of many drugs [e.g. morphine, paracetamol (acetaminophen)] but also capable of the biotransformation of important endogenous substrates (e.g. bilirubin, ethinylestradiol) and several xenobiotics. Isoform specificity for these substrates has, however, not been fully characterised. Serious adverse events associated with chloramphenicol toxicity in the neonate have highlighted the importance of developmental changes in UGT activity. However, isoform-specific differences preclude the generalisation of a simple developmental pattern for UGT activity. UGT2B7 is the only UGT isoform for which ontogeny has been characterised both in vitro and in vivo, using morphine as the probe drug. However, no general developmental pattern for the individual UGT isoforms which might be of value for the clinician is currently available. Genetic polymorphisms have been identified for the UGT family. Not only for the UGT1A gene, which reduces bilirubin glucuronidation, leading to genetic hyperbilirubinaemia (the Crigler-Najjar and Gilbert's syndromes), but also for 3 other UGT isoforms. However, the impact of these genetic differences on drug metabolism remains to be established because of overlapping isoform specificity of the drugs studied, as well as a lack of specific probe substrates to test the activity of individual UGT isoforms in relation to these gene mutations. Clearly, an information gap exists regarding the developmental and genetic aspects of UGT regulation and its potential impact on therapy. More research is needed on the pharmacogenetics and ontogeny of the UGTs for effective translation of scientific information into clinically applicable knowledge.

Ontogenic expression of natriuretic peptide mRNAs in postnatal rat brain: Implications for development?

The central natriuretic peptide system is composed of at least three structurally homologous and uniquely distributed peptides and receptors which are thought to be involved in the central regulation of cardiovascular and autonomic function and more recently been shown to affect cellular growth and proliferation, processes pertinent to mammalian development. As such, following our initial mapping of preproatrial natriuretic peptide (ppANP) mRNA in adult brain [M.C. Ryan, A.L. Gundlach, Anatomical localization of preproatrial natriuretic peptide mRNA in the rat brain by in situ hybridization histochemistry: Novel identification in olfactory regions, J. Comp. Neurol., 356 (1995) 168–182], it was of interest to determine the ontogenic expression of natriuretic peptide mRNAs in the developing rat brain. Using in situ hybridization histochemistry of specific [ 35S]- or [ 33P]-labeled oligonucleotides, ppANP and preproC-type natriuretic peptide (ppCNP) mRNAs were detected in the developing rat brain from postnatal day 4 to day 60 (adult). PpANP mRNA was observed in many hindbrain, but only some forebrain, regions at postnatal day 4. Regional differences in the temporal expression of ppANP mRNA were apparent with ppANP mRNA detected in the medial preoptic area, mammillary nuclei and medial habenular nucleus at postnatal day 4 and in other areas including the arcuate and dorsomedial hypothalamic nuclei and in olfactory and limbic regions at postnatal day 10. A number of regions also exhibited transient expression of ppANP mRNA such as the bed nucleus of the stria terminalis and the medial cerebellar nucleus. In contrast, ppCNP mRNA was detected at relatively high levels in several regions on postnatal day 4 including olfactory nuclei, the hippocampus and particularly the pontine nucleus. The level of expression appeared to increase markedly in most regions including forebrain olfactory and hippocampal areas and in brainstem regions including the pontine nucleus, the parvocellular and lateral reticular and spinal trigeminal nuclei by postnatal days 10 and 13, but decreased from this peak to equivalent to adult levels by postnatal day 28. The differential and transient expression of the natriuretic peptides during postnatal development, together with previous reports of the ontogenic regulation of natriuretic peptide receptor expression and binding patterns, further suggests their involvement in developmental processes in the rat CNS and provides information relevant to the likely functional development of natriuretic peptide-utilizing pathways.

Ovine glucose transporter-1 and -3: cDNA partial sequences and developmental gene expression in the placenta

Transplacental glucose transfer utilizes specific glucose transporter (GLUT) proteins. cDNAs encoding ovine placental GLUT1 and GLUT3 genes were isolated, cloned and sequenced and then used to investigate their developmental regulation in ovine placenta. A cDNA of approximately 2.2 kb was isolated from a Clontech lambda gt10 ovine adult liver cDNA library using a 436-bp rat GLUT1 cDNA probe. Sequence data obtained from this clone (1600 bp) demonstrated 97 per cent homology to nucleotides 477-2079 of bovine GLUT1. The deduced amino acid sequence of the ovine cDNA presented 99 per cent identity to amino acid 103-493 of bovine GLUT1, and 97-98 per cent identity to corresponding regions in human and rat GLUT1 deduced amino acid sequences. Reverse transcription-PCR (RT-PCR) was used to isolate an ovine cDNA fragment from placental total RNA. Forward and reverse primers (16 mer) were designed to amplify a predicted 483-bp fragment between the second transmembrane-spanning domain (M2) and intracellular loop of GLUT3. The deduced 161 amino acid sequence of the ovine cDNA demonstrated 84 and 88 per cent identity with murine and human GLUT3. These cDNAs were used to investigate the ontogeny of placental oGLUT1 and oGLUT3 gene expression by Northern analysis. Total RNA was extracted from ovine placenta at gestational days 45, 60, 90, 120 and 138 (n=6 per time point). Ovine GLUT1 gene expression increased significantly from days 45 to 60 (P<0.05), peaked at around day 120 of gestation, then decreased to about two-thirds of maximal levels by day 138 of gestation (term 147 +/- 2). Ovine GLUT3 gene expression increased throughout gestation with significant increases from days 45 to 60, 60 to 120 and 120 to 138 (P<0.05). This study confirms the presence of both GLUT1 and GLUT3 genes in the ovine placenta and demonstrates ontogenic regulation of gene expression. The difference in temporal gene expression between oGLUT1 and oGLUT3 suggests distinct roles for each transporter during development. The nucleotide sequences reported in this paper have been submitted to the GenBank/EMBL Data Bank under accession numbers U89029 (oGLUT1) and U89030 (oGLUT3).