Intrauterine growth restriction inhibits expression of eukaryotic elongation factor 2 kinase, a regulator of protein translation.

Nutrient deprivation suppresses protein synthesis by blocking peptide elongation. Transcriptional upregulation and activation of eukaryotic elongation factor 2 kinase (eEF2K) blocks peptide elongation by phosphorylating eukaryotic elongation factor 2. Previous studies examining placentas from intrauterine growth restricted (IUGR) newborn infants show decreased eEF2K expression and activity despite chronic nutrient deprivation. However, the effect of IUGR on hepatic eEF2K expression in the fetus is unknown. We, therefore, examined the transcriptional regulation of hepatic eEF2K gene expression in a Sprague-Dawley rat model of IUGR. We found decreased hepatic eEF2K mRNA and protein levels in IUGR offspring at birth compared with control, consistent with previous placental observations. Furthermore, the CpG island within the eEF2K promoter demonstrated increased methylation at a critical USF 1/2 transcription factor binding site. In vitro methylation of this binding site caused near complete loss of eEF2K promoter activity, designating this promoter as methylation sensitive. The eEF2K promotor in IUGR offspring also lost the protective histone covalent modifications associated with unmethylated CGIs. In addition, the +1 nucleosome was displaced 3' and RNA polymerase loading was reduced at the IUGR eEF2K promoter. Our findings provide evidence to explain why IUGR-induced chronic nutrient deprivation does not result in the upregulation of eEF2K gene transcription.

Intrauterine growth restriction disrupts developmental epigenetics around distal growth hormone response elements on the rat hepatic IGF-1 gene.

Intrauterine growth restriction (IUGR) decreases serum IGF-1 levels. Postnatal IGF-1 expression is transcriptionally regulated by growth hormone (GH) through growth hormone response elements (GHREs). We hypothesized that IUGR disrupts the normal developmental maturation of hepatic IGF-1 intron 2 growth hormone response element (IN2GHRE) histone methylation of key lysines and DNA methylation. We also evaluated a 5' distal weak enhancer (IGF-1 5'-upstream region growth hormone response element; 5URGHRE) as a GHRE specificity control. IUGR was induced through a well-characterized model of bilateral uterine artery ligation of the pregnant rat. Offspring livers were tested at d 0 and 21. Chromatin immunoprecipitation and bisulfite sequencing quantified epigenetic characteristics. We found that distinct age-related developmental patterns of histone and DNA methylation characterize each GHRE. Development increased H3K4 trimethylation (me3) in both GHREs. However, H3K9me3 decreased with age at IN2GHRE and increased with age at 5URGHRE. IUGR altered the developmental pattern of H3K4me3 and K9me3 around the GHREs in a sex-specific manner at d 21. Developmental and IUGR-induced DNA methylation occurred in a GHRE-, CpG site-, and sex-specific manner. We conclude that IUGR disrupts developmental epigenetics around distal GHREs on the rat hepatic IGF-1 gene.

Intrauterine growth restriction perturbs nucleosome depletion at a growth hormone-responsive element in the mouse IGF-1 gene.

Intrauterine growth restriction (IUGR) is a common human pregnancy complication. IUGR offspring carry significant postnatal risk for early-onset metabolic syndrome, which is associated with persistent reduction in IGF-1 protein expression. We have previously shown that preadolescent IUGR male mice have decreased hepatic IGF-1 mRNA and circulating IGF-1 protein at postnatal day 21, the age when growth hormone (GH) normally upregulates hepatic IGF-1 expression. Here we studied nucleosome occupancy and CpG methylation at a putative growth hormone-responsive element in intron 2 (in2GHRE) of the hepatic IGF-1 gene in normal, sham-operated, and IUGR mice. Nucleosome occupancy and CpG methylation were determined in embryonic stem cells (ESCs) and in liver at postnatal days 14, 21, and 42. For CpG methylation, additional time points out to 2 yr were analyzed. We confirmed the putative mouse in2GHRE was GH-responsive, and in normal mice, a single nucleosome was displaced from the hepatic in2GHRE by postnatal day 21, which exposed two STAT5b DNA binding sites. Nucleosome displacement correlated with developmentally programmed CpG demethylation. Finally, IUGR significantly altered the nucleosome-depleted region (NDR) at the in2GHRE of IGF-1 on postnatal day 21, with either complete absence of the NDR or with a shifted NDR exposing only one of two STAT5b DNA binding sites. An NDR shift was also seen in offspring of sham-operated mothers. We conclude that prenatal insult such as IUGR or anesthesia/surgery could perturb the proper formation of a well-positioned NDR at the mouse hepatic IGF-1 in2GHRE necessary for transitioning to an open chromatin state.

IUGR increases chromatin-remodeling factor Brg1 expression and binding to GR exon 1.7 promoter in newborn male rat hippocampus.

Intrauterine growth restriction (IUGR) increases the risk for neurodevelopment delay and neuroendocrine reprogramming in both humans and rats. Neuroendocrine reprogramming involves the glucocorticoid receptor (GR) gene that is epigenetically regulated in the hippocampus. Using a well-characterized rodent model, we have previously shown that IUGR increases GR exon 1.7 mRNA variant and total GR expressions in male rat pup hippocampus. Epigenetic regulation of GR transcription may involve chromatin remodeling of the GR gene. A key chromatin remodeler is Brahma-related gene-1(Brg1), a member of the ATP-dependent SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex. Brg1 regulates gene expression by affecting nucleosome repositioning and recruiting transcriptional components to target promoters. We hypothesized that IUGR would increase hippocampal Brg1 expression and binding to GR exon 1.7 promoter, as well as alter nucleosome positioning over GR promoters in newborn male pups. Further, we hypothesized that IUGR would lead to accumulation of specificity protein 1 (Sp1) and RNA pol II at GR exon 1.7 promoter. Indeed, we found that IUGR increased Brg1 expression and binding to GR exon 1.7 promoter. We also found that increased Brg1 binding to GR exon 1.7 promoter was associated with accumulation of Sp1 and RNA pol II carboxy terminal domain pSer-5 (a marker of active transcription). Furthermore, the transcription start site of GR exon 1.7 was located within a nucleosome-depleted region. We speculate that changes in hippocampal Brg1 expression mediate GR expression and subsequently trigger neuroendocrine reprogramming in male IUGR rats.

IUGR prevents IGF-1 upregulation in juvenile male mice by perturbing postnatal IGF-1 chromatin remodeling.

BACKGROUND: Intrauterine growth restriction (IUGR) offspring with rapid catch-up growth are at increased risk for early obesity especially in males. Persistent insulin-like growth factor-1 (IGF-1) reduction is an important risk factor. Using a mouse model of maternal hypertension-induced IUGR, we examined IGF-1 levels, promoter DNA methylation, and histone H3 covalent modifications at birth (D1). We additionally investigated whether prenatal perturbations could reset at preadolescence (D21). METHODS: IUGR was induced via maternal thromboxane A2-analog infusion in mice. RESULTS: IUGR uniformly decreased D1 IGF-1 mRNA and protein levels with reduced promoter 1 (P1) transcription and increased P1 DNA methylation. IUGR males also had increased H3K4ac at exon 5 and 3' distal UTR. At D21, IUGR males continued to have decreased IGF-1 levels, originating from both P1 and P2 with reduced 1A variant. IUGR males also had decreased activation mark of H3K4me3 at P1 compared with sham males. In contrast, D21 IUGR females normalized their IGF-1 levels, in association with an increased activation mark of H3K4me3 at P1 compared with sham females. CONCLUSION: IUGR uniformly affected D1 hepatic IGF-1 epigenetic modifications in both sexes. However, at preadolescence, IUGR males are unable to correct for the prenatal reduction possibly due to a more perturbed IGF-1 chromatin structure.

Combination of intrauterine growth restriction and a high-fat diet impairs cholesterol elimination in rats.

BACKGROUND: Intrauterine growth restriction (IUGR) increases the risk of adult-onset hypercholesterolemia. High-fat diet (HFD) consumption potentiates IUGR-induced increased cholesterol. Cholesterol is converted to bile acids by Cyp7a1 in preparation for excretion. We hypothesized that IUGR rats fed a HFD will have increased cholesterol, decreased Cyp7a1 protein levels, and decreased bile acids compared to control rats fed a HFD. METHODS: At day 21, IUGR and control pups were placed on one of three diets: a regular chow or one of two HFDs containing 1% or 2% cholesterol. Cholesterol levels and hepatic Cyp7a1 protein levels were quantified a postnatal week 28. RESULTS: Both HFDs increased serum cholesterol levels in control rats, and HFD fed IUGR rats had further increased serum cholesterol up to 35-fold. Both HFDs increased hepatic cholesterol levels, and IUGR further increased hepatic cholesterol levels up to fivefold. IUGR decreased hepatic Cyp7a1 protein up to 75%, and hepatic bile acids up to 54%. CONCLUSION: IUGR increased cholesterol and bile acids and decreased Cyp7a1 protein in rats fed a HFD without changing food intake. These findings suggest that IUGR increases the vulnerability of HFD fed rats to hypercholesterolemia via decreased cholesterol conversion to bile acids.

Intrauterine growth restriction transiently delays alveolar formation and disrupts retinoic acid receptor expression in the lung of female rat pups.

BACKGROUND: We showed that intrauterine growth restriction (IUGR) increases distal airspace wall thickness at birth (postnatal age 0; P0) in rat pups (saccular stage of lung development). However, that report did not assess whether the saccular phenotype persisted postnatally or occurred in males or females, nor did the report identify a potential molecular pathway for the saccular phenotype at P0. We hypothesized that IUGR persistently delays alveolar formation and disrupts retinoic acid receptor (RAR) mRNA and protein levels in the lung of rat pups in a postnatal age- and sex-specific manner. METHODS: IUGR was induced in pregnant rats by bilateral uterine artery ligation. Alveolar formation and expression of RARα, -ß, and -γ were quantified at P0, P6 (alveolar stage), and P21 (postalveolarization). RESULTS: IUGR increased distal airspace wall thickness in female pups at P0 only. IUGR did not affect male pups at any age. IUGR transiently increased lung RAR-ß protein abundance, which inhibits alveolar formation, at P0 in female pups. Serum retinol concentration was normal at all ages. CONCLUSION: IUGR alone is not sufficient to persistently delay postnatal alveolar formation or disrupt expression of RARs. We speculate that for IUGR to delay alveolar formation postnatally, a second insult is necessary.

IUGR decreases elastin mRNA expression in the developing rat lung and alters elastin content and lung compliance in the mature rat lung.

Complications of intrauterine growth restriction (IUGR) include increased pulmonary morbidities and impaired alveolar development. Normal alveolar development depends upon elastin expression and processing, as well as the formation and deposition of elastic fibers. This is true of the human and rat. In this study, we hypothesized that uteroplacental insufficiency (UPI)-induced IUGR decreases mRNA levels of elastin and genes required for elastin fiber synthesis and assembly, at birth (prealveolarization) and postnatal day 7 (midalveolarization) in the rat. We further hypothesized that this would be accompanied by reduced elastic fiber deposition and increased static compliance at postnatal day 21 (mature lung). We used a well characterized rat model of IUGR to test these hypotheses. IUGR decreases mRNA transcript levels of genes essential for elastic fiber formation, including elastin, at birth and day 7. In the day 21 lung, IUGR decreases elastic fiber deposition and increases static lung compliance. We conclude that IUGR decreases mRNA transcript levels of elastic fiber synthesis genes, before and during alveolarization leading to a reduced elastic fiber density and increased static lung compliance in the mature lung. We speculate that the mechanism by which IUGR predisposes to pulmonary disease may be via decreased lung elastic fiber deposition.

Intrauterine growth restriction affects hippocampal dual specificity phosphatase 5 gene expression and epigenetic characteristics.

Intrauterine growth retardation (IUGR) predisposes humans toward hippocampal morbidities, such as impaired learning and memory. Hippocampal dual specificity phosphatase 5 (DUSP5) may be involved in these morbidities because DUSP5 regulates extracellular signal-regulated kinase phosphorylation (Erk). In the rat, IUGR causes postnatal changes in hippocampal gene expression and epigenetic characteristics. However, the impact of IUGR upon hippocampal DUSP5 expression and epigenetic characteristics is not known. We therefore hypothesized that IUGR affects hippocampal 1) DUSP5 expression, DNA CpG methylation, and histone code, and 2) erk1/2 phosphorylation in a well-characterized rat model of IUGR. We found that IUGR significantly decreased DUSP5 expression in the day of life (DOL) 0 and 21 male rat, while decreasing only DUSP5 protein levels in the DOL21 female rat. Fluorescent in situ hybridization and immunohistochemistry analyses localized the changes in DUSP5 mRNA and protein, many of which occurred in the dentate gyrus. IUGR also caused sex-specific differences in DNA CpG methylation and histone code in two sites of the hippocampal DUSP5 gene, a 5'-flanking specificity protein-1 (SP1) site and exon 2. Finally, when IUGR decreased DUSP5 protein levels, Erk phosphorylation increased. We conclude that IUGR affects hippocampal DUSP5 expression and epigenetic characteristics in a sex-specific manner.

IUGR differentially alters MeCP2 expression and H3K9Me3 of the PPARγ gene in male and female rat lungs during alveolarization.

Intrauterine growth restriction (IUGR) increases the risk of postnatal lung disease, with males more affected. In rat lungs, IUGR impairs alveolarization in conjunction with altered expression of peroxisome proliferator-activated receptor gamma (PPARγ). In non-lung cells, PPARγ transcription is regulated in part by the epigenetic modifying enzyme, and the methyl CpG binding protein 2 (MeCP2). However, it is unknown if IUGR affects MeCP2 expression or its interaction with PPARγ in the rat lung during alveolarization. In this study, we hypothesized that the rat lung would be characterized by the presence of MeCP2 short and long mRNA transcripts, MeCP2 protein isoforms, and the MeCP2 regulatory micro RNA, miR132. We further hypothesized that IUGR would, in a gender-specific manner, alter the levels of MeCP2 components in association with changes in PPARγ mRNA, MeCP2 occupancy at the PPARγ promoters, and PPARγ histone 3 lysine 9 trimethylation (H3K9Me3). To test these hypotheses, we used a well-characterized rat model of uteroplacental insufficiency-induced IUGR. We demonstrated the presence of MeCP2 mRNA, protein, and miR132 in the rat lung throughout alveolarization. We also demonstrated that IUGR alters MeCP2 expression and its interaction with PPARγ in a gender-divergent manner. We conclude that IUGR induces gender-specific alterations in the epigenetic milieu in the rat lung. We speculate that in the IUGR rat lung, this altered epigenetic milieu may predispose to gender-specific alterations in alveolarization.

Intrauterine growth retardation affects expression and epigenetic characteristics of the rat hippocampal glucocorticoid receptor gene.

Studies in humans and rats suggest that intrauterine growth retardation (IUGR) permanently resets the hypothalamic-pituitary-adrenal (HPA) axis. HPA axis reprogramming may involve persistently altered expression of the hippocampal glucocorticoid receptor (hpGR), an important regulator of HPA axis reactivity. Persistent alteration of gene expression, long after the inciting event, is thought to be mediated by epigenetic mechanisms that affect mRNA and mRNA variant expression. GR mRNA variants in both humans and rats include eleven 5'-end variants and GRalpha, the predominant 3'-end variant. The 3'-end variants associated with glucocorticoid resistance in humans (GRbeta, GRgamma, GRA, and GRP) have not been reported in rats. We hypothesized that in the rat hippocampus IUGR would decrease total GR mRNA, increase GRbeta, GRgamma, GRA, and GRP, and affect epigenetics of the GR gene at birth (D0) and at 21 days of life (D21). IUGR increased hpGR and exon 1.7 hpGR mRNA in males at D0 and D21, associated with increased trimethyl H3/K4 at exon 1.7 at both time points. IUGR also increased hpGRgamma in males at D0 and D21, associated with increased acetyl H3/K9 at exon 3 at both time points. hpGRA increased in female IUGR rats at D0 and D21. In addition, our data support the existence of hpGRbeta and hpGRP in the rat. IUGR has sex-specific, persistent effects on GR expression and its histone code. We speculate that postnatal changes in hippocampal GR variant and total mRNA expression may underlie IUGR-associated HPA axis reprogramming.

Epigenetics of programmed obesity: alteration in IUGR rat hepatic IGF1 mRNA expression and histone structure in rapid vs. delayed postnatal catch-up growth.

Maternal food restriction (FR) during pregnancy results in intrauterine growth-restricted (IUGR) offspring that show rapid catch-up growth and develop metabolic syndrome and adult obesity. However, continued nutrient restriction during nursing delays catch-up growth and prevents development of obesity. Epigenetic regulation of IGF1, which modulates growth and is synthesized and secreted by the liver, may play a role in the development of these morbidities. Control (AdLib) pregnant rats received ad libitum food through gestation and lactation, and FR dams were exposed to 50% food restriction from days 10 to 21. FR pups were nursed by either ad libitum-fed control dams (FR/AdLib) or FR dams (FR/FR). All pups were weaned to ad libitum feed. Maternal FR resulted in IUGR newborns with significantly lower liver weight and, with the use of chromatin immunoprecipitation, decreased dimethylation at H3K4 in the IGF1 region was observed. Obese adult FR/AdLib males had decreased dimethylation and increased trimethylation of H3K4 in the IGF1 region. This corresponded to an increase in mRNA expression of IGF1-A (134 ± 5%), IGF1-B (165 ± 6%), IGF1 exon 1 (149 ± 6%), and IGF1 exon 2 (146 ± 7%) in the FR/AdLib compared with the AdLib/AdLib control group. In contrast, nonobese FR/FR had significantly higher IGF1-B mRNA levels (147 ± 19%) than controls with no difference in IGF1-A, exon 1 or exon 2. Modulation of the rate of IUGR newborn catch-up growth may thus protect against IGF1 epigenetic modifications and, consequently, obesity and associated metabolic abnormalities.

Fetal growth restriction alters transcription factor binding and epigenetic mechanisms of renal 11beta-hydroxysteroid dehydrogenase type 2 in a sex-specific manner.

Intrauterine growth restriction (IUGR) increases the risk of serious adult morbidities such as hypertension. In an IUGR rat model of hypertension, we reported a persistent decrease in kidney 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) mRNA and protein levels from birth through postnatal (P) day 21. This enzyme deficiency can lead to hypertension by limiting renal glucocorticoid deactivation. In the present study, we hypothesized that IUGR affects renal 11beta-HSD2 epigenetic determinants of chromatin structure and alters key transcription factor binding to the 11beta-HSD2 promoter in association with persistent downregulation of its mRNA expression. To test this hypothesis, we performed bilateral uterine artery ligation on embryonic day 19.5 pregnant rats and harvested kidneys at day 0 (P0) and P21. Key transcription factors that can affect 11beta-HSD2 expression include transcriptional enhancers specificity protein 1 (SP1) and NF-kappaB p65 and transcriptional repressors early growth response factor (Egr-1) and NF-kappaB p50. Our most important findings were as follows: 1) IUGR significantly decreased SP1 and NF-kappaB (p65) binding to the 11beta-HSD2 promoter in males, while it increased Egr-1 binding in females and NF-kappaB (p50) binding in males; 2) IUGR increased CpG methylation status, as well as modified the pattern of methylation in several CpG sites of 11beta-HSD2 promoter at P0 also in a sex-specific manner; and 3) IUGR decreased trimethylation of H3K36 in exon 5 of 11beta-HSD2 at P0 and P21 in both genders. We conclude that IUGR is associated with altered transcriptional repressor/activator binding in connection with increased methylation in the 11beta-HSD2 promoter region in a sex-specific manner, possibly leading to decreased transcriptional activity. Furthermore, IUGR decreased trimethylation of H3K36 of the 11beta-HSD2 gene in both genders, which is associated with decreased transcriptional elongation. We speculate that alterations in transcription factor binding and chromatin structure play a role in in utero reprogramming.

Epigenetics: intrauterine growth retardation (IUGR) modifies the histone code along the rat hepatic IGF-1 gene.

Intrauterine growth restriction (IUGR) decreases serum insulin growth factor-1 (IGF-1) levels. IGF-1 is an epigenetically regulated gene that has two promoters, alternative exon 5 splicing, and multiple termination sites. The regulation of gene expression involves the whole gene, as evidenced by the aforementioned IGF-1 paradigm. We hypothesized that IUGR in the rat would affect hepatic IGF-1 expression and alter the epigenetic characteristics of the IGF-1 gene along its length. IUGR was induced through a bilateral uterine artery ligation of the pregnant rat, a well-characterized model of IUGR. Pups from anesthesia and sham-operated dams were used as controls. Real-time RT-PCR and ELISA was used to measure expression at day of life (DOL) 0 and 21. Bisulfite sequencing and chromatin immunoprecipitation (ChIP) quantified IGF-1 epigenetic characteristics. A nontranscribed intergenic control was used for ChIP studies. IUGR decreased hepatic and serum IGF-1. Concurrently, IUGR modified epigenetic characteristics, particularly the histone code, along the length of the hepatic IGF-1 gene. Many changes persisted postnatally, and the postnatal effect of IUGR on the histone code was gender-specific. We conclude that IUGR modifies epigenetic characteristics of the rat hepatic IGF-1 gene along the length of the whole gene.

Growth retardation alters the epigenetic characteristics of hepatic dual specificity phosphatase 5.

Uteroplacental insufficiency leads to intrauterine growth retardation (IUGR) and adult onset insulin resistance in both humans and rats. IUGR rat liver is characterized by persistent changes in histone 3 lysine 9 and lysine 14 acetylation, which may induce postnatal changes in gene expression. We hypothesized that it would be possible to identify hepatic genes whose epigenetic characteristics and mRNA levels are altered due to IUGR using chromatin immunoprecipitation (ChIP) coupled with random primed differential display polymerase chain reaction (PCR). One of the isolated sequences identified contained exon 2 of the dual specificity phosphatase-5 gene (DUSP5). IUGR affected hepatic DUSP5 mRNA levels and exon 2 DNA methylation into adulthood in the rat. DUSP5 dephosphorylates Erk1 and Erk2 within the MAPK signaling cascade, which in turn affects serine 612 phosphorylation of insulin receptor substrate-1 (p612 IRS-1). In adult rat liver, IUGR increased Erk1/Erk2 phosphorylation and p612 IRS-1 phosphorylation. Increased serine phosphorylation of hepatic IRS-1 may contribute to the insulin resistance that characterizes these animals. We conclude that intrauterine growth retardation induced by uteroplacental insufficiency 1) affects the hepatic epigenetic characteristics and mRNA of the DUSP-5 and 2) increases hepatic insulin receptor substrate-1 phosphorylation at serine 612 in adult rats.

Uteroplacental insufficiency induces site-specific changes in histone H3 covalent modifications and affects DNA-histone H3 positioning in day 0 IUGR rat liver.

Uteroplacental insufficiency and subsequent intrauterine growth retardation (IUGR) increase the risk of adult onset insulin resistance and dyslipidemia in humans and rats. IUGR rats are further characterized by postnatal alterations in hepatic PPAR-gamma coactivator (PGC-1) and carnitine-palmitoyl-transferase I (CPTI) expression, as well as overall hyperacetylation of histone H3. However, it is unknown whether the histone H3 hyperacetylation is site specific or relates to the changes in gene expression previously described in IUGR rats. We therefore hypothesized that uteroplacental insufficiency causes site-specific modifications in hepatic H3 acetylation and affects the association of acetylated histone H3 with PGC-1 and CPTI promoter sequences. Uteroplacental insufficiency was used to produce asymmetrical IUGR rats. IUGR significantly increased acetylation of H3 lysine-9 (H3/K9), lysine-14 (H3/K14), and lysine-18 (H3/K18) at day 0 of life, and these changes occurred in association with decreased nuclear protein levels of histone deacetylase 1 (HDAC1) and HDAC activity. Chromatin immunoprecipitation using acetyl-H3/K9 antibody and day 0 chromatin revealed that uteroplacental insufficiency affected the association between acetylated H3/K9 and the promoters of PGC-1 and CPTI, respectively, in IUGR liver. At day 21 of life, the neonatal pattern of H3 hyperacetylation persisted only in the IUGR males. We conclude that uteroplacental insufficiency increases H3 acetylation in a site-specific manner in IUGR liver and that these changes persist in male IUGR animals. The altered association of the PGC-1 and CPTI promoters with acetylated H3/K9 correlates with previous reports of IUGR altering the expression of these genes. We speculate that in utero alterations of chromatin structure contribute to fetal programming.

Maternal Hyperglycemia Disrupts Histone 3 Lysine 36 Trimethylation of the IGF-1 Gene.

In utero environmental adaptation may predispose to lifelong morbidity. Organisms fine-tune gene expression to achieve environmental adaptation by epigenetic alterations of histone markers of gene accessibility. One example of epigenetics is how uteroplacental insufficiency-induced intrauterine growth restriction (IUGR), which predisposes to adult onset insulin resistance, decreases postnatal IGF-1 mRNA variants and the gene elongation mark histone 3 trimethylation of lysine 36 of the IGF-1 gene (H3Me3K36). Limitations in the study of epigenetics exist due to lack of a primary transgenic epigenetic model. Therefore we examined the epigenetic profile of insulin-like growth factor 1 (IGF-1) in a well-characterized rat model of maternal hyperglycemia to determine if the epigenetic profile of IGF-1 is conserved in disparate models of in utero adaptation. We hypothesized that maternal hyperglycemia would increase IGF-1 mRNA variants and H3Me3K36. However maternal hyperglycemia decreased hepatic IGF-1 mRNA variants and H3Me3K36. This finding is intriguing given that despite different prenatal insults and growth, both maternal hyperglycemia and IUGR predispose to adult onset insulin resistance. We speculate that H3Me3K36 of the IGF-1 gene is sensitive to the glucose level of the prenatal environment, with resultant alteration of IGF-1 mRNA expression and ultimately vulnerability to adult onset insulin resistance.

Uteroplacental insufficiency increases visceral adiposity and visceral adipose PPARgamma2 expression in male rat offspring prior to the onset of obesity.

Uteroplacental insufficiency (UPI) induced intrauterine growth restriction (IUGR) predisposes individuals to adult onset metabolic morbidities, including insulin resistance and cardiovascular disease. An underlying component of the development of these morbidities is adipose dysfunction; specifically a disproportionately abundant visceral adipose tissue. We hypothesize that IUGR will increase rats visceral adiposity and visceral expression of PPARgamma, a key regulator of adipogenesis. To test this hypothesis we employed a well described UPI induced IUGR rat model. Subcutaneous and visceral adipose levels were measured in adolescent control and IUGR rats using MRI. Expression of PPARgamma mRNA and protein, as well as PPARgamma target genes, was measured in neonatal, adolescent and adult rats. UPI induced IUGR increases the relative amount of visceral adipose tissue in male, but not female, adolescent rats in conjunction with an increase in PPARgamma2mRNA and protein in male visceral adipose. Importantly, these effects are seen prior to the onset of overt obesity. We conclude that increased PPARgamma2 expression in VAT of IUGR males is associated with increased visceral adiposity. We speculate that the increase in visceral adiposity may contribute to the metabolic morbidities experienced by this population.

IUGR decreases PPARγ and SETD8 Expression in neonatal rat lung and these effects are ameliorated by maternal DHA supplementation.

Intrauterine growth restriction (IUGR) is associated with altered lung development in human and rat. The transcription factor PPARγ, is thought to contribute to lung development. PPARγ is activated by docosahexanoic acid (DHA). One contribution of PPARγ to lung development may be its direct regulation of chromatin modifying enzymes, such as Setd8. In this study, we hypothesized that IUGR would result in a gender-specific reduction in PPARγ, Setd8 and associated H4K20Me levels in the neonatal rat lung. Because DHA activates PPARγ, we also hypothesized that maternal DHA supplementation would normalize PPARγ, Setd8, and H4K20Me levels in the IUGR rat lung. We found that IUGR decreased PPARγ levels, with an associated decrease in Setd8 levels in both male and female rat lungs. Levels of the Setd8-dependent histone modification, H4K20Me, were reduced on the PPARγ gene in both males and females while whole lung H4K20Me was only reduced in male lung. Maternal DHA supplementation ameliorated these effects in offspring. We conclude that IUGR decreases lung PPARγ, Setd8 and PPARγ H4K20Me independent of gender, while decreasing whole lung H4K20Me in males only. These outcomes are offset by maternal DHA. We speculate that maintenance of the epigenetic milieu may be one role of PPARγ in the lung and suggests a novel benefit of maternal DHA supplementation in IUGR.

Intrauterine growth restriction due to uteroplacental insufficiency decreased white matter and altered NMDAR subunit composition in juvenile rat hippocampi.

Uteroplacental insufficiency (UPI), the major cause of intrauterine growth restriction (IUGR) in developed nations, predisposes to learning impairment. The underlying mechanism is unknown. Neuronal N-methyl-d-aspartate receptors (NMDARs) are critical for synaptogenesis and learning throughout life. We hypothesized that UPI-induced IUGR alters rat hippocampal NMDAR NR2A/NR2B subunit ratio and/or NR1 mRNA isoform expression and synaptic density at day 21 (P21). To test this hypothesis, IUGR was induced by bilateral uterine artery ligation of the late-gestation Sprague-Dawley dam. At P21, hippocampal NMDAR subunit mRNA and protein were measured, as were levels of synaptophysin. Neuronal, synaptic, and glial density in CA1, CA3, and dentate gyrus (DG) was assessed by immunofluorescence. IUGR increased NR1 mRNA isoform NR1-3a and 1-3b expression in both sexes. In P21 males, IUGR increased protein levels of NR1 C2' and decreased NR1 C2, NR2A, and the NR2A-to-NR2B ratio, whereas in females, IUGR increased NR2B protein. In males, IUGR was associated with decreased myelin basic protein-to-neuronal nuclei ratio in CA1, CA3, and DG. We conclude that IUGR has sex-specific effects and that neither neuronal loss nor decreased synaptic density appears to account for the changes in NMDAR subunits. Rather, it is possible that synaptic NMDAR subunit composition is altered. Our results suggest that apparent recovery in the IUGR hippocampus may be associated with synaptic hyperexcitability. We speculate that the NMDAR plays an important role in IUGR-associated cognitive impairment.