Programmed regulation of rat offspring adipogenic transcription factor (PPARγ) by maternal nutrition.

We determined the protein expression of adipogenic transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ) and its co-repressor and co-activator complexes in adipose tissue from the obese offspring of under- and over-nourished dams. Female rats were fed either a high-fat (60% kcal) or control (10% kcal) diet before mating, and throughout pregnancy and lactation (Mat-OB). Additional dams were 50% food-restricted from pregnancy day 10 to term [intrauterine growth-restricted (IUGR)]. Adipose tissue protein expression was analyzed in newborn and adult male offspring. Normal birth weight Mat-OB and low birth weight IUGR newborns had upregulated PPARγ with variable changes in co-repressors and co-activators. As obese adults, Mat-OB and IUGR offspring had increased PPARγ with decreased co-repressor and increased co-activator expression. Nutritionally programmed increased PPARγ expression is associated with altered expression of its co-regulators in the newborn and adult offspring. Functional studies of PPARγ co-regulators are necessary to establish their role in PPARγ-mediated programmed obesity.

Tactile and Kinesthetic Stimulation (TKS) intervention improves outcomes in weanling rat bone in a neonatal stress model.

OBJECTIVES: Preterm infants are born with low bone mineral. Neonatal stress further impedes bone mineralization. Clinical evidence suggests that tactile and kinesthetic stimulation (TKS) improves bone phenotype and decreases stress response. Clinical and translational studies indicate the IGF-1 axis, responsible for postnatal growth and bone mineralization, is a key player. We hypothesized that TKS would attenuate the negative impact of neonatal stress on bone phenotype and the IGF-1 axis in weanling rats. METHODS: Neonatal stress (STRESS) or TKS (STRESS + 10min TKS) was administered from D6 to D10. Control animals received standard care. Tissue was harvested on D21. Dual energy x-ray absorptiometry (DXA) and bone morphometry were performed and serum osteocalcin, type I procollagen N-terminal propeptide (PINP), tartrate-resistant acid phosphatase (TRAP), and bone and liver mRNA levels of IGF-1, IGF-1 receptor (IGF-1R), and growth hormone receptor (GHR) were measured. RESULTS: Neonatal stress increased bone mineral content (BMC), area (BA), growth plate width, liver IGF-1 mRNA, and serum IGF-1. TKS maintained areal bone mineral density (aBMD) and bone specific IGF-1 and IGF-1R mRNA while STRESS decreased compared to controls. CONCLUSIONS: Neonatal stress results in apparent accelerated growth response. TKS differed from STRESS with improved tibia aBMD and increased bone specific IGF-1 mRNA.

Altered expression and chromatin structure of the hippocampal IGF1r gene is associated with impaired hippocampal function in the adult IUGR male rat.

Exposure to intrauterine growth restriction (IUGR) is an important risk factor for impaired learning and memory, particularly in males. Although the basis of IUGR-associated learning and memory dysfunction is unknown, potential molecular participants may be insulin-like growth factor 1 (Igf1) and its receptor, IGF1r. We hypothesized that transcript levels and protein abundance of Igf1 and IGF1r in the hippocampus, a brain region critical for learning and memory, would be lower in IUGR male rats than in age-matched male controls at birth (postnatal day 0, P0), at weaning (P21) and adulthood (P120). We also hypothesized that changes in messenger Ribonucleic acid (mRNA) transcript levels and protein abundance would be associated with specific histone marks in IUGR male rats. Lastly, we hypothesized that IUGR male rats would perform poorer on tests of hippocampal function at P120. IUGR was induced by bilateral ligation of the uterine arteries in pregnant dams at embryonic day 19 (term is 21 days). Hippocampal Igf1 mRNA transcript levels and protein abundance were unchanged in IUGR male rats at P0, P21 or P120. At P0 and P120, IGF1r expression was increased in IUGR male rats. At P21, IGF1r expression was decreased in IUGR male rats. Increased IGF1r expression was associated with more histone 3 lysine 4 dimethylation (H3K4Me2) in the promoter region. In addition, IUGR male rats performed poorer on intermediate-term spatial working memory testing at P120. We speculate that altered IGF1r expression in the hippocampus of IUGR male rats may play a role in learning and memory dysfunction later in life.

Mechanical-tactile stimulation (MTS) intervention in a neonatal stress model improves long-term outcomes on bone.

OBJECTIVES: Neonatal stress impairs postnatal bone mineralization. Evidence suggests that mechanical tactile stimulation (MTS) in early life decreases stress hormones and improves bone mineralization. Insulin-like growth factor (IGF1) is impacted by stress and essential to bone development. We hypothesized that MTS administered during neonatal stress would improve bone phenotype in later life. We also predicted an increase in bone specific mRNA expression of IGF1 related pathways. METHODS: Neonatal stress (STRESS) and MTS (STRESS+10 min of MTS) were given from D6 to D10 of rat life and tissue was harvested on D60 of life. Dual energy x-ray absorptiometry (DXA), bone morphometry, serum osteocalcin, type I procollagen N-terminal propeptide (PINP), tartrate-resistant acid phosphatase (TRAP), and bone and liver mRNA levels of IGF1, IGF1 receptor (IGF1R), and growth hormone receptor (GHR) were measured. RESULTS: Stress resulted in reduced bone area and bone mineral content (BMC) compared to naive control (CTL). MTS intervention increased BMC and tibial growth plate width compared to STRESS. MTS also resulted in higher osteocalcin, and, in males, lower TRAP (p<0.05). MTS resulted in three-fold, two-fold, and six-fold higher bone specific IGF1, IGF1R, and GHR, respectively (p ≤ 0.001) compared to STRESS. CONCLUSIONS: MTS in early postnatal life improves long-term bone mineralization. IGF1 and related pathways may explain improved BMC.

Epigenetics and fetal adaptation to perinatal events: diversity through fidelity.

Perinatal insults, including fetal undernutrition and hypoxia, are associated with an increased susceptibility to several adult-onset metabolic disorders. These include cardiovascular disease, insulin resistance, and obesity. However, the mechanisms driving the long-term phenotypic consequences have only recently begun to be elucidated. A primary mechanism accounting for perinatal adaptation is the epigenetic modification of chromatin. In this context, epigenetic modifications to chromatin are thought to arise in response to a perinatal insult in an effort to modulate gene expression and maximize fetal survival. In this symposium report, we discuss epigenetics as a mechanism by which perinatal adaptations can be made by the developing fetus. We examine the benefits of using multiple in vivo models to understand the interrelation of signals that come together and result in perinatal adaptation. Epigenetic effects on IGF-1 arising from a perinatal insult are discussed, as are the difficulties and challenges associated with this complex field. In conclusion, epigenetics provides a means of modulating gene transcription, thus allowing fetal adaptation to a broad variety of conditions.

Uteroplacental insufficiency decreases p53 serine-15 phosphorylation in term IUGR rat lungs.

Intrauterine growth restriction (IUGR) increases the incidence of chronic lung disease (CLD). The molecular mechanisms responsible for IUGR-induced acute lung injury that predispose the IUGR infant to CLD are unknown. p53, a transcription factor, plays a pivotal role in determining cellular response to stress by affecting apoptosis, cell cycle regulation, and angiogenesis, processes required for thinning of lung mesenchyme. Because thickened lung mesenchyme is characteristic of CLD, we hypothesized that IUGR-induced changes in lung growth are associated with alterations in p53 expression and/or modification. We induced IUGR through bilateral uterine artery ligation of pregnant rats. Uteroplacental insufficiency significantly decreased serine-15-phosphorylated (serine-15P) p53, an active form of p53, in IUGR rat lung. Moreover, we found that decreased phosphorylation of lung p53 serine-15 localized to thickened distal air space mesenchyme. We also found that IUGR significantly decreased mRNA for targets downstream of p53, specifically, proapoptotic Bax and Apaf, as well as Gadd45, involved in growth arrest, and Tsp-1, involved in angiogenesis. Furthermore, we found that IUGR significantly increased mRNA for Bcl-2, an antiapoptotic gene downregulated by p53. We conclude that in IUGR rats, uteroplacental insufficiency induces decreased lung mesenchymal p53 serine-15P in association with distal lung mesenchymal thickening. We speculate that decreased p53 serine-15P in IUGR rat lungs alters lung phenotype, making the IUGR lung more susceptible to subsequent injury.

Basic and translational research in neonatal pharmacology.

Pharmacologic study is needed in the extremely immature newborns who currently survive. Study is needed of both the drug treatment previously established in more mature neonates and of novel drug therapy. Carefully controlled studies are needed to identify accurately both beneficial and harmful drug therapy and the mechanisms of that toxicity. Careful pharmacologic study of drug disposition and its mechanisms might lead to dosing paradigms or patient selection that minimize toxicity and maximize efficacy. In vivo, translational models of neonatal diseases are limited, but can be used to identify novel treatments and study mechanisms of established, successful therapy. Findings from such studies can generate hypotheses for study in humans leading to a continuing scientific interchange from bedside to bench to bedside. Similarly, clinical observations can generate hypotheses for study in translational models where more invasive analyses are possible. Specific areas of drug treatment should focus on neonatal disorders with long-term, adverse outcomes, such as chronic lung disease, that is amenable to translational study with animal models. National data show a progressive decrease in the clinician-scientist pool entering biomedical research. The future of neonatal pharmacology studies requires an increase in training programs for the physician-scientist whose clinical education in neonatology can be complemented by rigorous basic-science training. Success as a clinician-scientist will require collaboration with full-time basic scientists who can continue studies during periods of clinical work and provide critical study methodology to the overall study design. Such a work environment must be supported by academic institutions and may require more flexibility in the promotion and tenure schedule and process, such as the nature of what it rewards. To complement this, the NIH could modify its grant reporting process to identify co-investigators in studies who may provide unique input to the study concepts and design, such as clinical correlations or clinical investigations.

Uteroplacental insufficiency affects epigenetic determinants of chromatin structure in brains of neonatal and juvenile IUGR rats.

Intrauterine growth retardation (IUGR) increases the risk of neuroendocrine reprogramming. In the rat, IUGR leads to persistent changes in cerebral mRNA levels. This suggests lasting alterations in IUGR cerebral transcriptional regulation, which may result from changes in chromatin structure. Candidate nutritional triggers for these changes include altered cerebral zinc and one-carbon metabolite levels. We hypothesized that IUGR affects cerebral chromatin structure in neonatal and postnatal rat brains. Rats were rendered IUGR by bilateral uterine artery ligation; controls (Con) underwent sham surgery. At day of life 0 (d0), we measured cerebral DNA methylation, histone acetylation, expression of chromatin-affecting enzymes, and cerebral levels of one-carbon metabolites and zinc. At day of life 21 (d21), we measured cerebral DNA methylation and histone acetylation, as well as the caloric content of Con and IUGR rat breast milk. At d0, IUGR significantly decreased genome-wide and CpG island methylation, as well as increased histone 3 lysine 9 (H3/K9) and histone 3 lysine 14 (H3/K14) acetylation in the hippocampus and periventricular white matter, respectively. IUGR also decreased expression of the chromatin-affecting enzymes DNA methyltransferase 1 (DNMT1), methyl-CpG binding protein 2 (MeCP2), and histone deacetylase (HDAC)1 in association with increased cerebral levels of zinc. In d21 female IUGR rats, cerebral CpG DNA methylation remained lower, whereas H3/K9 and H3/K14 hyperacetylation persisted in hippocampus and white matter, respectively. In d21 male rats, IUGR decreased acetylation of H3/K9 and H3/K14 in these respective regions compared with controls. Despite these differences, caloric, fat, and protein content were similar in breast milk from Con and IUGR dams. We conclude that IUGR results in postnatal changes in cerebral chromatin structure and that these changes are sex specific.

Evidence of the effect of 'specialization' on the management, surgical outcome and survival from colorectal cancer in Wessex.

BACKGROUND: A prospective audit of the management of colorectal cancer was established to investigate factors associated with variation in survival observed within the former Wessex region (population three million). METHODS: Some 5173 patients (4562 surgically treated) with colorectal cancer diagnosed between 1991 and 1994 were followed for 5 years. Details of referral, diagnosis, surgical treatment, postoperative complications and outcomes were collected. The association between surgical outcomes and survival and both case volume and specialization (defined to include membership of the Association of Coloproctology of Great Britain and Ireland) was explored, accounting for variables with prognostic significance. RESULTS: There was a statistically significant association between high-volume operators (more than 50 operations per year) and specialization. The greatest benefit was observed with respect to specialists versus non-specialists, in terms of a lower postoperative mortality rate (odds ratio 0.67 (95 per cent confidence interval (c.i. 0.53 to 0.84)), lower anastomotic leak rates (odds ratio 0.46 (c.i. 0.31 to 0.66)), higher local recurrence-free survival (hazard ratio 0.56 (0.44 to 0.71)) and better long-term survival (hazard ratio 0.76 (c.i. 0.71 to 0.83)). CONCLUSION: There is a stronger association between surgical specialization in coloproctology and beneficial outcome than with high-volume caseloads. This is not entirely accounted for by case-mix or patient population, and is seen following colonic and rectal surgery and among patients with advanced disease.

Uteroplacental insufficiency alters liver and skeletal muscle branched-chain amino acid metabolism in intrauterine growth-restricted fetal rats.

Uteroplacental insufficiency causes intrauterine growth restriction (IUGR) and decreases plasma levels of the branched-chain amino acids in both humans and rats. Increased fetal oxidation of these amino acids may contribute to their decline in the IUGR fetus. The rate-limiting step of branched-chain amino acid oxidation is performed by the mitochondrial enzyme branched-chain alpha-keto acid dehydrogenase (BCKAD), which is regulated by a deactivating kinase. We therefore hypothesized that uteroplacental insufficiency increases BCKAD activity through altered mRNA and protein levels of BCKAD and/or the BCKAD kinase. In IUGR fetal liver, BCKAD activity was increased 3-fold, though no difference in hepatic BCKAD protein or mRNA levels were noted. Hepatic BCKAD kinase mRNA and protein levels were significantly decreased in association with the increase in BCKAD activity. In IUGR fetal skeletal muscle, BCKAD mRNA levels were significantly increased. IUGR skeletal muscle BCKAD protein levels as well as BCKAD kinase mRNA and protein levels were unchanged. We also quantified mRNA levels of two amino acid transporters: LAT1 (system L) and rBAT (cysteine and dibasic amino acids). Both hepatic and muscle LAT1 mRNA levels were significantly increased in the IUGR fetus. We conclude that uteroplacental insufficiency significantly increases hepatic BCKAD activity in association with significantly decreased mRNA and protein levels of the deactivating kinase. We speculate that these changes contribute to the decreased serum levels of branched-chain amino acids seen in the IUGR fetus and may be an adaptation to the deprived milieu associated with uteroplacental insufficiency.

Altered expression and function of mitochondrial beta-oxidation enzymes in juvenile intrauterine-growth-retarded rat skeletal muscle.

Uteroplacental insufficiency and subsequent intrauterine growth retardation (IUGR) affects postnatal metabolism. In juvenile rats, IUGR alters skeletal muscle mitochondrial gene expression and reduces mitochondrial NAD(+)/NADH ratios, both of which affect beta-oxidation flux. We therefore hypothesized that gene expression and function of mitochondrial beta-oxidation enzymes would be altered in juvenile IUGR skeletal muscle. To test this hypothesis, mRNA levels of five key mitochondrial enzymes (carnitine palmitoyltransferase I, trifunctional protein of beta-oxidation, uncoupling protein-3, isocitrate dehydrogenase, and mitochondrial malate dehydrogenase) and intramuscular triglycerides were quantified in 21-d-old (preweaning) IUGR and control rat skeletal muscle. In isolated skeletal muscle mitochondria, enzyme function of the trifunctional protein of beta-oxidation and isocitrate dehydrogenase were measured because both enzymes compete for mitochondrial NAD(+). Carnitine palmitoyltransferase I, the trifunctional protein of beta-oxidation, and uncoupling protein 3 mRNA levels were significantly increased in IUGR skeletal muscle, whereas mRNA levels of isocitrate dehydrogenase and mitochondrial malate dehydrogenase were unchanged. Similarly, trifunctional protein of beta-oxidation activity was increased in IUGR skeletal muscle mitochondria, and isocitrate dehydrogenase activity was unchanged. Interestingly, skeletal muscle triglycerides were significantly increased in IUGR skeletal muscle. We conclude that uteroplacental insufficiency alters IUGR skeletal muscle mitochondrial lipid metabolism, and we speculate that the changes observed in this study play a role in the long-term morbidity associated with IUGR.

Myocardial gene expression of glucose transporter 1 and glucose transporter 4 in response to uteroplacental insufficiency in the rat.

Uteroplacental insufficiency causes intrauterine growth retardation (IUGR) and subsequent low birth weight, which predisposes the affected newborn towards adult Syndrome X. Individuals with Syndrome X suffer increased morbidity from adult ischemic heart disease. Myocardial ischemia initiates a defensive increase in cardiac glucose metabolism, and individuals with Syndrome X demonstrate reduced insulin sensitivity and reduced glucose uptake. Glucose transporters GLUT1 and GLUT4 facilitate glucose uptake across cardiac plasma membranes, and hexokinase II (HKII) is the predominant hexokinase isoform in adult cardiac tissue. We therefore hypothesized that GLUT1, GLUT4 and HKII gene expression would be reduced in heart muscle of growth-retarded rats, and that reduced gene expression would result in reduced myocardial glucose uptake. To prove this hypothesis, we measured cardiac GLUT1 and GLUT4 mRNA and protein in control IUGR rat hearts at day 21 and at day 120 of life. HKII mRNA quantification and 2-deoxyglucose-uptake studies were performed in day-120 control and IUGR cardiac muscle. Both GLUT1 and GLUT4 mRNA and protein were significantly reduced at day 21 and at day 120 of life in IUGR hearts. HKII mRNA was also reduced at day 120. Similarly, both basal and insulin-stimulated glucose uptake were significantly reduced in day-120 IUGR cardiac muscle. We conclude that adult rats showing IUGR as a result of uteroplacental insufficiency express significantly less cardiac GLUT1 and GLUT4 mRNA and protein than control animals (which underwent sham operations), and that this decrease in gene expression occurs in parallel with reduced myocardial glucose uptake. We speculate that this reduced GLUT gene expression and glucose uptake contribute towards mortality from ischemic heart disease seen in adults born with IUGR.

Uteroplacental insufficiency lowers the threshold towards hypoxia-induced cerebral apoptosis in growth-retarded fetal rats.

Infants suffering uteroplacental insufficiency and hypoxic ischemic injury often demonstrate cerebral apoptosis. Our objective was to determine the global effects of uteroplacental insufficiency upon cerebral gene expression of the apoptosis related proteins Bcl-2 and Bax and their role in increasing vulnerability to hypoxia-induced cerebral apoptosis. We therefore caused uteroplacental insufficiency and growth retardation by performing bilateral uterine artery ligation upon pregnant rats 2 days prior to term delivery and elicited further perinatal fetal hypoxia by placing maternal rats in 14% FiO(2) 3 h prior to delivery. We quantified cerebral levels of Bcl-2 and Bax mRNA, lipid peroxidation, caspase-3 activity, and cAMP in control and growth retarded term rat pups that experienced either normoxia or hypoxia. Uteroplacental insufficiency alone caused a significant decrease in cerebral Bcl-2 mRNA levels without altering cerebral Bax mRNA levels, malondialdehyde levels, or caspase-3 activity. In contrast, uteroplacental insufficiency and subsequent fetal hypoxia significantly increased cerebral Bax mRNA levels, lipid peroxidation and caspase-3 activity; Bcl-2 mRNA levels continued to be decreased. Hypoxia alone increased cerebral cAMP levels, whereas uteroplacental insufficiency and subsequent hypoxia decreased cerebral cAMP levels. We speculate that the decrease in Bcl-2 gene expression increases the vulnerability towards cerebral apoptosis in fetal rats exposed initially to uteroplacental insufficiency and subsequent hypoxic stress.

Uteroplacental insufficiency alters hepatic fatty acid-metabolizing enzymes in juvenile and adult rats.

Multiple adult morbidities are associated with intrauterine growth retardation (IUGR) including dyslipidemia. We hypothesized that uteroplacental insufficiency and subsequent IUGR in the rat would lead to altered hepatic fatty acid metabolism. To test this hypothesis, we quantified hepatic mRNA levels of acetyl-CoA carboxylase (ACC), carnitine palmitoyltransferase (CPTI), the beta-oxidation-trifunctional protein (HADH), fasting serum triglycerides, and hepatic malonyl-CoA levels at different ages in control and IUGR rats. Fetal gene expression of all three enzymes was decreased. Juvenile gene expression of CPTI and HADH continued to be decreased, whereas gene expression of ACC was increased. Serum triglycerides were unchanged. A sex-specific response was noted in the adult rats. In males, serum triglycerides, hepatic malonyl-CoA levels, and ACC mRNA levels were significantly increased, and CPTI and HADH mRNA levels were significantly decreased. In contrast, the female rats demonstrated no significant changes in these variables. These results suggest that uteroplacental insufficiency leads to altered hepatic fatty acid metabolism that may contribute to the adult dyslipidemia associated with low birth weight.

Uteroplacental insufficiency alters cerebral mitochondrial gene expression and DNA in fetal and juvenile rats.

Uteroplacental insufficiency increases the risk of perinatal and long-term neurologic morbidity by depriving the fetus of oxidative substrate and causing intrauterine growth retardation. Skeletal muscle and liver from growth retarded fetal and juvenile rats respond to this deprivation by altering mitochondrial gene expression and function. The objective of this study was to determine whether cerebral mitochondrial mRNA is similarly altered in fetal and juvenile growth retarded rats and to correlate these alterations with mitochondrial DNA and marker protein levels. To fulfill this objective, mRNA levels of four important mitochondrial proteins were quantified using RT-PCR in growth retarded and sham-operated control fetal and juvenile rat brains; these proteins were NADH-ubiquinone oxireductase subunit 4, subunit C of the F1F0-ATPase, and the adenine nucleotide transporters 1 and 2. Mitochondrial DNA/nuclear DNA ratios and mitochondrial 60 kD marker protein levels were also quantified in growth retarded and sham-operated control fetal and juvenile rat brains using PCR and Western Blotting, respectively. Cerebral mRNA levels of all four proteins were increased in the IUGR fetuses and decreased in the IUGR juvenile animals. Cerebral mitochondrial/nuclear DNA ratios and mitochondrial marker protein levels were not significantly altered in the IUGR fetuses; however, both were significantly diminished in IUGR juvenile pups. These studies suggest that the metabolic stresses associated with uteroplacental insufficiency in the rat cause altered fetal and postnatal cerebral mitochondrial mRNA and DNA levels.

Placental expression of glucose transporter proteins 1 and 3 in growth-restricted fetal rats.

OBJECTIVE: The purpose of this study was to determine the effect of intrauterine growth restriction on the placental expression of glucose transporter proteins. STUDY DESIGN: Intrauterine growth restriction was induced by bilateral uterine artery ligation in the pregnant rat at a gestational age of 19 days (term is 21.5 days). Maternal rats were killed and fetuses were delivered by hysterotomy on gestational days 20 and 21. Control fetuses from mothers that had been subjected to a sham operation were studied simultaneously. Glucose transporter protein 1 and glucose transporter protein 3 messenger ribonucleic acid was quantified by reverse transcriptase-polymerase chain reaction amplification. Glucose transporter protein 1 and glucose transporter protein 3 densities in placental membranes were also assessed by Western blotting and by immunohistochemical analysis. RESULTS: Glucose transporter protein 1 messenger ribonucleic acid, expressed as a multiple of the matched sham control value, was unchanged on both days 20 and 21 of gestation. Glucose transporter protein 3 messenger ribonucleic acid was also unchanged. Western blotting demonstrated no change in expression of glucose transporter protein 1 or glucose transporter protein 3 on either day 20 or 21 of gestation. Immunohistochemical staining patterns for glucose transporter protein 1 and glucose transporter protein 3 on the syncytiotrophoblastic membranes were similar between the growth-restricted group and the sham control group. CONCLUSION: Placental expression of glucose transporter proteins in the pregnant rat is unchanged with uteroplacental insufficiency.

Localization and quantification of glucose transporters in liver of growth-retarded fetal and neonatal rats.

To determine whether altered transport of glucose into the hepatocyte may be an important factor contributing to abnormal hepatic glucose metabolism in the intrauterine growth-retarded (IUGR) fetus and newborn, we measured glucose transport (glucose uptake, GLUT protein, and mRNA) and localization of GLUT protein in liver of control (sham operated) and IUGR fetal (day 20) and postnatal (1, 4, 14, and 21 days) rats. GLUT-1 and -2 proteins were localized to the hepatocyte. Glucose uptake and GLUT-1 protein and mRNA levels were increased in IUGR fetal and neonatal liver. GLUT-2 protein and mRNA levels were low in IUGR and control fetal liver. After birth, GLUT-2 abundance did not differ from controls. Run-on experiments showed that the rate of transcription of GLUT-1 and -2 did not differ between IUGR and control rats. However, the transcription rate of GLUT-1 decreased with age, and the GLUT-2 transcription rate increased with age. These studies indicate that the metabolic and physiological factors that cause IUGR also alter glucose transporter expression in fetal liver.

Intrauterine growth retardation alters mitochondrial gene expression and function in fetal and juvenile rat skeletal muscle.

Uteroplacental insufficiency alters the anabolic metabolism of the fetus, resulting in intrauterine growth retardation (IUGR). The metabolic and physiologic factors that cause IUGR have long standing consequences after birth. Postnatal growth and glucose metabolism are altered in the IUGR infant. Skeletal muscle is an important component of growth and metabolizes up to 70% of i.v. glucose. The ability of skeletal muscle to metabolize glucose is affected by ATP availability. We hypothesized that gene expression and function of proteins involved in mitochondrial ATP production and distribution would be altered in juvenile IUGR muscle. To test this hypothesis, we used a model of IUGR, induced by bilateral uterine artery ligation in the pregnant rat, that mimics uteroplacental insufficiency in the human. RT-PCR was used to measure the mRNA levels of three important mitochondrial proteins; NADH-ubiquinone-oxireductase subunit 4L(ND-4L), subunit C of the F1F0-ATP synthase (SUC), and adenine nucleotide translocator 1 (ANT1) in IUGR and control rats in fetal and juvenile life. In the fetus, mRNA levels of all three proteins were significantly increased in IUGR skeletal muscle. In contrast, in juvenile animals, mRNA levels of all three proteins were significantly decreased. mRNA levels of other metabolically important proteins, glucose-6-phosphate dehydrogenase and carnitine-palmitoyl-transferase II, were not significantly altered in IUGR juvenile animals. To assess if decreased gene expression is associated with altered mitochondrial function, we measured the mitochondrial NAD+/NADH ratio in d 21 juvenile control and IUGR muscle. At d 21, decreased gene expression if ND-4L, SUC, and ANTI is associated with a decreased mitochondrial NAD+/NADH ratio. The results of our study suggest that the metabolic alterations associated with uteroplacental insufficiency in the rat result in altered fetal and postnatal muscle mitochondrial mRNA expression as well as altered postnatal mitochondrial function.

Measurement of GLUT mRNA in liver of fetal and neonatal rats using a novel method of quantitative polymerase chain reaction.

Transfer of glucose into the hepatocyte is mediated by glucose transporters (GLUTs). GLUT mRNA levels are usually measured by Northern blot analysis. Reverse transcription-polymerase chain reaction (RT-PCR) is often used to measure RNA abundance. However, this method is only semiquantitative and has no internal control during first-strand synthesis. We designed a method of coreverse transcription and PCR amplification using bovine rhodopsin as an internal control for both cDNA synthesis and amplification. As part of the validation of this technique, we determined that there was no nonspecific amplification of bovine GLUTs by rhodopsin primers, that there were no differences in amplification due to different regions of the Glut gene amplified, and that there were no secondary structure effects on amplification. We applied our modified method of RT-PCR to measure the ontogeny of GLUT expression in liver of fetal and postnatal rats (d20 fetuses and d1, d4, d14, and d21 juvenile rat pups). GLUT 1 mRNA quantity decreased whereas GLUT 2 increased with age. We were able to detect small quantities of GLUT 3 in fetal liver and of GLUT 5 in postnatal liver. This method of RT-PCR provides an internal control and allows measurement of mRNA levels in small quantities of tissue, making it ideal for use in the fetus and any system in which mRNA levels are low.

Altered hepatic gene expression of enzymes involved in energy metabolism in the growth-retarded fetal rat.

Intrauterine growth retardation (IUGR) resulting from placental insufficiency is a common complication of pregnancy. Bilateral uterine artery ligation of the pregnant rat is a model which mimics intrauterine growth retardation in the human. IUGR rat fetuses have altered hepatic energy and redox states, with reduced fetal hepatic ATP/ADP ratio, increased cytosolic NAD+/NADH ratio, and decreased mitochondrial NAD+/NADH ratio. These critical changes in energy metabolism contribute to IUGR. The effects of these changes at the molecular level are largely unknown. To address these effects we compared hepatic mRNA populations of IUGR and normal fetuses and neonates using mRNA differential display, a polymerase chain reaction-based method for assaying transcriptional differences under various conditions. We isolated and sequenced 18 cDNA products whose mRNA levels were elevated in IUGR compared with normal fetal and neonatal liver. These analyses demonstrated that NADH-ubiquinone oxireductase subunit 4L mRNA (ND-4L) was significantly increased in liver of IUGR fetuses and neonates. This suggested that IUGR may be associated with altered expression of genes involved in the generation of ATP and NADH. Therefore, we measured mRNA levels of adenine-nucleotide translocator-2 (ANT-2), glucose-6-phosphate dehydrogenase (G6PD), mitochondrial malate dehydrogenase (MMD), ornithine transcarbamylase (OTC), and phosphofructokinase-2 (PFK-2) using a semiquantitative reverse transcriptase-polymerase chain reaction-based technique. In the IUGR fetus, ND-4L, ANT-2, G6PD, and MMD mRNA levels were significantly elevated; PFK-2 mRNA levels were unchanged, and OTC levels were decreased. In the IUGR newborn rat, mRNA levels of all 6 enzymes were increased suggesting that the metabolic state of the growth retarded newborn remains abnormal after birth. Uteroplacental insufficiency affects the immediate and long-term metabolic milieu of the growth retarded animal, and forces specific adjustments, including the expression of mRNA encoding enzymes involved with hepatic energy production.