Curr Diab Rep (2012) 12:67–74 DOI 10.1007/s11892-011-0248-1

DIABETES AND PREGNANCY (CJ HOMKO, SECTION EDITOR)

Metabolic Programming, Epigenetics, and Gestational Diabetes Mellitus Sara E. Pinney & Rebecca A. Simmons

Published online: 30 November 2011 # Springer Science+Business Media, LLC 2011

Abstract The link between an adverse intrauterine environment and the development of disease later in life has been observed in offspring of pregnancies complicated by obesity and diabetes, but the molecular mechanisms underlying this phenomenon are unknown. In this review, we highlight recent publications exploring the role of gestational diabetes mellitus in the programming of disease in the offspring. We also review recent publications aiming to identify mechanisms responsible for the “programming effect” that results from exposure to diabetes in utero. Finally, we highlight research on the role of epigenetic regulation of gene expression in an animal model of uteroplacental insufficiency where the offspring develop diabetes as a model by which an exposure to the mother can alter epigenetic modifications that affect expression of key genes and ultimately lead to the development of diabetes in the offspring. Keywords Gestational diabetes mellitus . Type 2 diabetes . Intrauterine growth retardation . Metabolic programming . Histone modifications . DNA methylation . Epigenetics . β-cell . Exendin-4 . Oxidative stress . Obesity

S. E. Pinney (*) Division of Endocrinology and Diabetes, Department of Pediatrics, The Children’s Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, 3400 Civic Center Boulevard, Philadelphia, PA 19104, USA e-mail: [email protected] R. A. Simmons Division of Neonatology, Department of Pediatrics, The Children’s Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, 415 Curie Boulevard, Philadelphia, PA 19104, USA e-mail: [email protected]

Introduction Diabetic pregnancy induces marked abnormalities in glucose homeostasis and insulin secretion in the fetus that result in abnormal fetal growth [1]. Population-based studies have demonstrated that the offspring of diabetic mothers have an increased risk for obesity, glucose intolerance, and type 2 diabetes mellitus (T2DM) [2–5]. Individuals whose cord blood or amniotic fluid insulin levels were elevated have a three- to fourfold risk for developing glucose intolerance, obesity, and T2DM in late childhood [5]. It has been proposed that early exposure to elevated insulin levels may lead to a malprogramming of critical functions related to the development of diabetes and obesity later in life [6]. Theories exploring the fetal basis of adult disease have evolved in recent years, and stem from the idea that environmental factors in early life and in utero can have profound influences on lifelong health [7, 8]. Epidemiologic and animal studies by a number of investigators support the concept that there is a critical developmental window of programming in which in utero or neonatal exposures or events can make an individual more susceptible to the development of adult diseases such as obesity and diabetes [9]. The link between an adverse intrauterine environment and the development of disease later in life has been observed in offspring of pregnancies complicated by obesity and diabetes, but the molecular mechanisms underlying this phenomenon are unknown [10]. Exposure to an adverse intrauterine milieu, including the environment of hyperglycemia and hyperinsulinemia, may disturb epigenetic, structural, and functional adaptive responses responsible for developmental programming. In this review, we highlight recent publications exploring the role of gestational diabetes mellitus (GDM) in the programming of disease in the offspring. We also review recent publications aiming to identify mechanisms responsible for

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the “programming effect” that results from exposure to diabetes mellitus in utero. Finally, we highlight our research on the role of epigenetic regulation of gene expression in an animal model of uteroplacental insufficiency where the offspring develop diabetes as adults.

GDM and Metabolic Programming of the Offspring: Recent Epidemiologic Studies Two recent studies examined the relationship between maternal glucose concentrations at 28 weeks gestation and measurements of early childhood obesity. Pettitt et al. [11•] evaluated the relationship of glycemic levels during pregnancy with anthropometry in offspring of diabetic and nondiabetic pregnant women from the United Kingdom as part of the multinational HAPO (Hyperglycemia and Adverse Pregnancy Outcome) study. The authors found that there was a significant association with maternal 1-hour blood sugars during gestational diabetes testing and offspring body mass index (BMI) z score≥85th percentile at 2 years of age (P=0.017), but other relationships between the offspring’s measurements of obesity and maternal glucose levels were indistinguishable from offspring of control women at this age. The authors plan to continue to follow this cohort as the offspring age. A second study examined the relationship between maternal glucose concentrations among women without pre-existing diabetes or GDM diagnosis and BMI of their offspring at age 3 years [12•]. In their adjusted model, when compared with glucose concentrations less than 100 mg/dL, a maternal glucose concentration≥130 mg/dL was associated with approximately a twofold greater risk of childhood overweight/ obesity with an adjusted risk ratio of 2.34 (95% CI,1.25– 4.36), after adjusting for maternal prepregnancy BMI. The authors conclude that fetal exposure to high maternal glucose concentrations in the absence of pre-existing diabetes or GDM may contribute to the development of overweight/obesity in the offspring, independent of maternal prepregnancy BMI [12•]. Tsadok, et al. [13] examined the outcomes of obesity and hypertension in 17-year-old offspring born to mothers with GDM in pregnancy. This study utilized the Jerusalem Perinatal Study birth cohort containing 92,408 birth records collected from 1964 to 1976. Follow-up data on offspring BMI and blood pressure were obtained from Israeli military records. The cohort contained 293 women with GDM and 59,499 control women without recorded GDM. The authors report that after adjusting for birth weight, GDM remained significantly associated with offspring BMI and diastolic blood pressure at age 17. However, when the subcohort (women enrolled from 1974 to 1976, who self-reported prepregnancy BMI) was adjusted with prepregnancy BMI,

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the association between GDM and offspring BMI was attenuated. The authors conclude that maternal characteristics have long-term effects on cardiometabolic outcomes of their offspring at age 17 years. Some limitations of this study were the lack of routine GDM screening in Israel at the time of data collection and that the prevalence of GDM in the cohort (0.5%) was lower than the current prevalence of 3% to 5%. In addition, the authors did not have any data about the severity of the GDM and the outcome data was only available for 65% of the offspring (38% of the females and 82% of the males). Furthermore, the prepregnancy BMI in the subcohort was self-reported. Crume et al. [14] examined the association between exposure to maternal diabetes in utero and BMI growth trajectories from birth through age 13 years. The authors used a mixed linear effects model to assess difference in BMI and BMI growth velocity from birth through 13 years of age for 95 subjects exposed to diabetes in utero and 409 unexposed subjects enrolled in a retrospective cohort study. They found that the overall BMI growth trajectory (adjusted for sex and race/ethnicity) was not significantly different for exposed and unexposed subjects from birth through 26 months of age (P=0.48). However, the overall BMI growth trajectory from 27 months through 13 years was significantly greater in the offspring exposed to diabetes in utero after adjusting for sex and race/ethnicity. This difference was primarily due to a significantly higher BMI growth velocity among exposed youth between 10 and 13 years, increasing by 4.56 kg/m2 in the exposed group compared with an increase of 3.51 kg/m2 in the unexposed group. A limitation of this study is that the authors did not include pubertal status in the model and did not assess whether advanced pubertal stage may have accounted for the increased BMI velocity. The authors controlled for demographic variables, socioeconomic factors, and prepregnancy BMI, which did not alter the observed associations. In summary, the epidemiologic studies from 2010 to 2011 support previous studies finding an association between exposure to diabetes in pregnancy and adverse outcomes in the offspring, indicating that the in utero exposure effectively programs the individual to develop various diseases, including obesity and hypertension, later in life.

Mechanisms by Which Exposure to Gestational Diabetes Programs the Offspring for Future Development of Disease Many researchers are exploring mechanisms that may be responsible for the programming of diabetes and obesity in the offspring born to mothers with gestational diabetes. Steculorum et al. [15•] recently published a manuscript exploring the consequences of maternal diabetes on the

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organization of hypothalamic neural circuits that are involved in the regulation of energy balances. For this work, the authors used a mouse model of insulin deficiency created via streptozotocin injections on gestational day 5.5. The authors report that maternal diabetes was associated with changes in growth as revealed by a significantly higher pre- and post-weaning body weight in the offspring of insulin-deficient dams relative to those of control mice. Mice born to diabetic dams also had increased fasting glucose levels, increased insulin levels, and increased food intake during their adult life. These impairments in metabolic regulation were associated with leptin resistance in adulthood. The ability of leptin to activate the intracellular signaling in the arcuate nucleus was also decreased in diabetic dams. Neural projections from the arcuate nucleus to the paraventricular nucleus were markedly reduced in the offspring of insulin-deficient dams. These data show that animals born to diabetic dams display abnormally organized hypothalamic feeding pathways that could result from attenuated responsiveness of hypothalamic neurons to the neurotropic actions of leptin during neonatal development. Thus, insulin deficiency and hyperglycemia during pregnancy have long-term consequences for metabolic regulation. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that regulate metabolic anti-inflammatory and developmental processes. There are three subtypes of PPARs including PPAR-α, PPAR-δ, and PPAR-γ. In maternal diabetes, the excess of glucose and other metabolic substrates such as lipids in the maternal circulation reaches intrauterine tissues and generates a proinflammatory environment that impairs embryonic, fetal, and placental development [16]. In this context, PPARs arise as regulator of signaling pathways that could help to prevent impairments in intrauterine development that are induced by maternal diabetes. Holdsworth-Carson et al. [17] recently published a study examining PPARs and their role in the pathologies of the human placenta in GDM, intrauterine growth retardation (IUGR), and preeclampsia (PE). They obtained human placenta at term from women with GDM, and compared it with uncomplicated term placentae. They also examined placentae from women with offspring exposed to IUGR, PE, or both. The authors found that GDM was associated with significantly lower PPAR-γ mRNA and protein, and PPAR-α protein and retinoid X receptor-α (RXR-α; the binding partner for PPARs) protein, whereas PPAR binding activity remained unchanged. Placentae from women with PE did not demonstrate any changes in mRNA or protein expression or PPAR DNA binding activity whereas placentae from women with IUGR/PE showed significant increases in PPAR-α protein, PPAR-γ mRNA and protein, and RXRαmRNA and protein expression. Significantly elevated protein expression of PPAR-α and RXR-α were associated with IUGR placentae. IUGR and

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IUGR/PE-exposed placentae had significantly higher PPAR-γ DNA binding activity compared with controls. In summary, the paper suggests that PPARs may be involved in the pathophysiology of GDM, IUGR, and PE. Diminished concentrations of various prostaglandins PGE2 and PGI2 have been found in embryos from diabetic rats and PGI2 is capable of increasing embryonic PGE2 concentrations through the activation of the nuclear receptor PPAR-δ [18]. PPAR-δ activators are lipid molecules such as oleic and linoleic acids that are present in high levels of safflower oil and olive oil, respectively. The aim of the study published by Higa et al. [18] was to analyze the capability of dietary supplements from either 6% olive or 6% safflower oils, to regulate PGE2, PGI2, and nitric oxide concentration in embryo and deciduas from control and diabetic rats early in organogenesis. Diabetes was induced by a single injection of streptozotocin 1 week before mating. Animals were fed oil-supplemented diets from gestational day 0.5 to 10.5. The authors reports that olive oil– and safflower oil– supplemented treatments highly reduced rates of fetal resorption and malformations in diabetic animals and that the oils were able to prevents maternal diabetes-induced alterations in embryonic and decidual PGI2 and PGE2 concentrations. Dietary treatments of olive oil or safflower oil also prevented nitric oxide overproduction in embryos and deciduas from diabetic rats. Finally, Pavlinkova et al. [19••] hypothesized that the developmental defects in the diabetic embryo are due to alterations of critical development pathways that may arise as a result of altered gene expression when compared with control embryos. Pavlinkova et al. [19••] reported the results from gene expression profiling of embryos exposed to diabetes in pregnancy and controls. When comparing gene expression profiles with normal embryos at midgestation, the authors found significantly altered gene expression in the diabetes-exposed embryos at gestational day 10.5. They identified 126 genes with expression levels that were more than twofold changed and 83% of those genes had decreased expression (twofold or greater) when compared with controls. The largest group of genes affected included transcription factors, which function as DNAbinding molecules that affect transcriptional regulation. The authors examined the promoters of the 126 genes that had differential expression compared with controls and after searching for transcription factor binding motifs, they found a diverse set of conserved motifs in the 5′ upstream region of the differentially expressed genes. They found an overrepresentation of the binding site motifs for FOX01, FOX04, and NRF2, which are genes known to be associated with oxidative stress. These findings are consistent with the idea that embryos exposed to maternal diabetes have altered gene expression, which plays an

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important role in the pathogenesis of diabetes-induced programming of adult disease. The changes in gene expression in the diabetes-exposed embryo appear to be permanent and thus indicate that these changes are induced by epigenetic modifications, which could include histone modifications and/or DNA methylation. To our knowledge, there have not been any studies to date that describe particular changes in DNA methylation or specific histone modifications in embryonic tissue exposed to diabetes in pregnancy or more specifically GDM, which ultimately lead to changes in gene expression. Therefore, the following sections describe specific epigenetic modifications induced in an IUGR animal model and their relationship to the development of T2DM in the offspring. This example serves as a model by which an exposure to the mother can alter epigenetic modifications that affect expression of key genes that ultimately can lead to the development of diabetes in the offspring.

Chromatin Structure, DNA Methylation, and Gene Expression Epigenetic modifications of the genome provide a mechanism that allows the stable propagation of gene expression from one generation of cells to the next [20–23]. Epigenetic states can be modified by environmental factors, which may contribute to the development of abnormal phenotypes. There are at least three distinct categories through which epigenetic information can be inherited: histone modifications, DNA methylation, and noncoding RNAs. Histone Modifications In eukaryotes, the nucleosome is formed when DNA is wrapped around an octameric complex of two molecules of each of the four histones: H2A, H2B, H3, and H4. The amino termini of histones can be modified by acetylation, methylation, sumoylation, phosphorylation, glycosylation, and ADP ribosylation. The most common histone modifications involve acetylation and methylation of lysine residues in the amino termini of H3 and H4. Increased acetylation induces transcription activation, whereas decreased acetylation usually induces transcription repression. Methylation of histones, conversely, is associated with both transcription activation and repression. Moreover, lysine residues can be mono-, di-, or trimethylated in vivo, providing an additional mechanism of regulation [23]. DNA Methylation The second class of epigenetic regulation is DNA methylation, in which a cytosine base is modified by a DNA

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methyltransferase at the C5 position of cytosine, a reaction that is carried out by various members of a single family of enzymes. Approximately 70% of CpG dinucleotides in human DNA are constitutively methylated, whereas most of the unmethylated CpGs are located in CpG islands. CpG islands are CG-rich sequences located near coding sequences, and serve as promoters for their associated genes. Approximately half of mammalian genes have CpG islands. The methylation status of CpG islands within promoter sequences works as an essential regulatory element by modifying the binding affinity of transcription factors to DNA binding sites. In normal cells, most CpG islands remain unmethylated; however, under circumstances such as cancer [24–27] and oxidative stress, they can become methylated de novo. This aberrant methylation is accompanied by local changes in histone modification and chromatin structure, such that the CpG island and its embedded promoter take on a repressed conformation that is incompatible with gene transcription. It is not known why particular CpG islands are susceptible to aberrant methylation. DNA methylation is erased in the early embryo and then re-established at the time of implantation [28, 29], although the purpose of this process is not known. Differential DNA methylation is established through two opposing mechanisms: first, through the wave of de novo methylation at the time of blastocyst implantation and second, through a mechanism that protects CpG islands from DNA methylation. The specifics of these mechanisms have yet to be elucidated [30]. DNA methylation is commonly associated with gene silencing and contributes to X-chromosomal inactivation, genomic imprinting, as well as transcriptional regulation of tissue-specific genes during cellular differentiation [31, 32]. Histone methylation can affect DNA methylation patterns and vice versa [30]. For example, methylation of lysine 9 on histone 3 (H3) promotes DNA methylation, whereas CpG methylation stimulates methylation of lysine 9 on H3 [32]. Recent evidence indicates that this dual relationship between histone methylation and DNA methylation might be accomplished by direct interactions between histone and DNA methyltransferases [30]. Thus, chromatin modifications induced by adverse stimuli are self-reinforcing and can propagate. Noncoding RNAs New evidence from a variety of model systems demonstrates that noncoding RNAs, such as microRNAs, small RNAs, and long or large RNAs, play a significant role in epigenetic gene regulation and chromosomal dynamics including mechanisms for processes such as dosage compensation, imprinting and gene silencing by RNA

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interference [33]. Noncoding RNAs with different regulatory functions are a common feature of mammalian transcriptomes, especially after the discovery that most of the eukaryotic genomes are transcribed into RNAs that have no protein-coding potential [34]. Noncoding RNAs are able to direct the cytosine methylation and histone modifications that are related to gene expression regulation in complex organisms in addition to having several other unrelated functions [33, 35]. Recent reports suggest that gene silencing, mediated by DNA methylation, can be induced by promoter-directed silencing RNAs (siRNAs) in mammalian cells [33, 36, 37]. Kawasaki et al. [35] found that siRNAs targeted to a promoter could induce H3K9 methylation in various mammalian cell lines. It is becoming clear that these molecules are very important in various epigenetic modification mechanisms such as heterochromatin silencing, transposon activity and silencing, and X chromosome inactivation; however, much remains to be learned about their specific epigenetic roles.

Epigenetic Regulation of Gene Expression in Fetal Growth Retardation A number of studies suggest that uteroplacental insufficiency, the most common cause of IUGR in the developed world, induces epigenetic modifications in offspring [7, 38–40]. Fetal growth retardation can be induced by bilateral uterine artery ligation in the pregnant rat [7, 8]. Following ligation, pups are born spontaneously and have decreased levels of glucose, insulin, insulin growth factor-1, and amino acids [7]. At birth, their body weights are decreased compared with control, but they have increased fat mass by 2 weeks of age and by 26 weeks of age the fat pad mass of the IUGR animals is 1.8 times greater than controls [7]. In this model, diabetes develops in animals at approximately 15 to 26 weeks of age with underlying β-cell secretory defects and insulin resistance, the salient features of most forms T2DM in humans [7, 8]. Epigenetic modifications affecting processes important to glucose regulation and insulin secretion, characteristics essential to the pathophysiology of T2DM, have been described in the IUGR pancreatic β cells and muscle. Although no specific experiments have looked at the epigenetic control of gene expression in offspring exposed to GDM specifically, the data presented here suggest that in the IUGR model, epigenetic regulation of gene expression plays an important role in the development of adult disease. The following sections describe specific epigenetic modifications induced in the IUGR model and their relationship to the development of T2DM.

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Chromatin Remodeling in the β-Cell of IUGR Rats Pdx1 is a homeodomain-containing transcription factor that plays a critical role in the early development of both the endocrine and exocrine pancreas, and in the later differentiation and function of the β cell. As early as 24 h after the onset of growth retardation, Pdx1 mRNA levels are reduced by more than 50% in IUGR fetal rats. Suppression of Pdx1 expression persists after birth and progressively declines in the IUGR animal, implicating an epigenetic mechanism. A change in histone acetylation is the first epigenetic modification found in β cells of IUGR animals. Islets isolated from IUGR fetuses show a significant decrease in H3 and H4 acetylation at the proximal promoter of Pdx1 [39]. These changes in H3 and H4 acetylation are associated with a loss of binding of USF1 to the proximal promoter of Pdx1 [39]. USF1 is a critical activator of Pdx1 transcription, and its decreased binding markedly decreases Pdx1 transcription [41, 42]. After birth, histone deacetylation progresses and is followed by a marked decrease in H3K4 trimethylation and a significant increase in dimethylation of H3K9 in IUGR islets [39]. H3K4 trimethylation is usually associated with active gene transcription whereas H3K9 dimethylation is usually a repressive chromatin mark. Progression of these histone modifications parallels the progressive decrease in Pdx1 expression that manifests as defective glucose homeostasis and increased oxidative stress in the aging IUGR animals [39]. Nevertheless, at 2 weeks of age, the silencing histone modifications in the IUGR pup are responsible for suppression of Pdx1 expression since there is no appreciable methylation of CpG islands in mice at this age [39]. Reversal of histone deacetylation in IUGR islets at 2 weeks of age is sufficient to nearly normalize Pdx1 mRNA levels permanently, perhaps due to active β-cell replication present in the neonatal rodent [39]. In IUGR, Pdx1 is first silenced due to recruitment of corepressors, including histone deacetylase 1 (HDAC1) and mSin3A [21]. These repressors catalyze histone deacetylation. Binding of these deacetylases facilitates loss of trimethylation of H3K4, further repressing Pdx1 expression [39]. We found that inhibition of HDAC activity by trichostatin A treatment normalizes H3K4me3 levels at Pdx1 in IUGR islets [39]. These data suggest that the association of HDAC1 at Pdx1 in IUGR islets likely serves as a platform for the recruitment of a demethylase, which catalyzes demethylation of H3K4. The molecular mechanism responsible for DNA methylation in IUGR islets is likely dependent on the methylation status of lysine 9 on H3 (H3K9). Previous studies have shown that changes in methylation of H3K9 precede changes in DNA methylation [43, 44]. It has also been suggested that

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Fig. 1 A short course of treatment with exendin-4 during the newborn period leads to increased USF1 binding at the proximal Pdx1 promoter leading to increased HAT activity and increased PCAF binding. Binding of these factors results in increased histone H3 Ac and increased methylation of H3K4 in addition to the prevention of DNA methylation. These epigenetic modifications result in increased

transcription of Pdx-1 and ultimately prevention of the development of diabetes in the IUGR model. A Compacted chromatin and transcriptional silencing of Pdx1 in IUGR islets. B Relaxed chromatin and transcriptional activation of Pdx1 in IUGR islets treated with exendin-4. HAT—histone acetyl transferase; H3 Ac—H3 acetylation; IUGR—intrauterine growth retardation

DNA methyltransferases may act only on chromatin that is methylated at H3K9 [45]. Another class of enzymes (eg, DNA methyltransferase 3A [DNMT3A] and DNA methyltransferase 3B ([DNMT3B]) bind to DNA methylases to initiate DNA methylation [40]). These results demonstrate that IUGR induces a selfpropagating epigenetic cycle in which the mSin3A/HDAC complex is first recruited to the Pdx1 promoter, histone tails are subjected to deacetylation, and Pdx1 transcription is repressed. At the neonatal stage, this epigenetic process is reversible and may define an important developmental window for therapeutic approaches. However, as dimethylated H3K9 accumulates, DNMT3A is recruited to the promoter and initiates de novo DNA methylation, which locks in the silenced state in the IUGR adult pancreas resulting in diabetes. How do these epigenetic events lead to diabetes? Targeted homozygous disruption of Pdx1 in mice results in pancreatic agenesis, and homozygous mutations yield a similar phenotype in humans [46]. Milder reductions in Pdx1 protein levels, as occurs in the Pdx+/− mice, allow for the development of a normal mass of β cells [46], but result in the impairment of several events in glucose-stimulated insulin secretion [46]. These results indicate that Pdx1 plays a critical role in the normal function of β cells [46] in addition to its role in β-cell lineage development. This may be the reason that humans with heterozygous missense mutations in Pdx1 exhibit early and late onset forms of T2DM [46]. Exendin-4 (Ex-4), a long-acting glucagon-like peptide-1 analogue, given in the newborn period increases Pdx1

expression and prevents the development of diabetes in the IUGR rat [8, 47••]. We found that phosphorylation of USF1 was markedly increased in IUGR islets in Ex-4–treated animals and this resulted in increased USF1 and PCAF association at the proximal promoter of Pdx1, thereby increasing histone acetyl transferase (HAT) activity (Fig. 1) [47••]. Histone H3 acetylation and trimethylation of H3K4 were permanently increased, whereas subsequent DNA methylation was prevented at the proximal promoter of Pdx1 in IUGR islets [47••]. In summary, normalization of these epigenetic modifications reversed silencing of Pdx1 in islets of IUGR animals. These studies demonstrate a novel mechanism whereby a short treatment course of Ex-4 in the newborn period permanently increases HAT activity by recruiting USF1 and PCAF to the proximal promoter of Pdx1, which restores chromatin structure at the Pdx1 promoter and prevents DNA methylation, thus preserving Pdx1 transcription (Fig. 1) [47••].

Conclusions The discovery of a theoretical time period during which aberrant epigenetic modifications may be reversed represents a therapeutic window for the use of novel agents that could prevent common diseases with late-onset phenotypes [8, 47••]. T2DM and perhaps obesity are such diseases, where predisposed individuals could be treated with agents that normalize the epigenetic programming of key genes, thus providing protection against development of the adult diseased phenotype.

Curr Diab Rep (2012) 12:67–74 Disclosure No potential conflicts of interest relevant to this article were reported.

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