Initiation of Metformin in Early Pregnancy Results in Fetal Bioaccumulation, Growth Restriction & Renal Dysmorphology in a Primate Model.

BACKGROUND: In recent years, pragmatic metformin use in pregnancy has stretched to include prediabetes, type 2 diabetes, gestational diabetes and (most recently) pre-eclampsia. With its expanded use, however, concerns of unintended harm have been raised. OBJECTIVE: We developed an experimental primate model and applied triple-quadruple pole LC mass spectrometry (UHPLC-QQQ) for direct quantitation of maternal and fetal tissue metformin levels with detailed fetal biometry and histopathology. STUDY DESIGN: Within 30 days of confirmed conception (defined as early pregnancy), n=13 time-bred (TMB) Rhesus dams with gestations designated for fetal necropsy were initiated on twice daily human dose-equivalent 10 mg/kg metformin or vehicle control. Pregnant dams were maintained as pairs and fed either a control chow or 36% fat Western-style diet (WSD). Metformin or placebo vehicle control were delivered in a variety of treats while animals were separated via a slide. A Cesarean was performed at G145, and amniotic fluid and blood were collected and the fetus and placenta were delivered. The fetus was immediately necropsied by trained primate center personnel. All fetal organs were dissected, measured, sectioned, and processed per clinical standards. Fluid and tissue metformin levels were assayed using validated UHPLC-QQQ in SRM against standard curves. RESULTS: Among the n=13 G145 pregnancies with fetal necropsy, n=1 dam and its fetal tissues had detectable metformin levels despite being allocated to the vehicle control group (>1 µM metformin/kg maternal weight or fetal/placental tissue), while a second fetus allocated to the vehicle control group had severe fetal growth restriction (birthweight 248.32 g, <1%) and was suspected of having a fetal congenital condition. After excluding these two fetal gestations from further analyses, 11 fetuses from dams initiated on either vehicle control (n=4, 3 female, 1 male fetuses) or 10 mg/kg metformin (n=7, 5 female, 2 male fetuses) were available for analyses. Among dams initiated on metformin by G30 (regardless of maternal diet), we observed significant bioaccumulation within the fetal kidney (0.78-6.06 µmol/kg, mean 2.48 µmol/kg) , liver (0.16-0.73 µmol/kg, mean 0.38 µmol/kg), fetal gut (0.28-1.22 µmol/kg, mean 0.70 µmol/kg), amniotic fluid (0.43-3.33 µmol/L, mean 1.88 µmol/L), placenta (0.16-1.0 µmol/kg , mean 0.50 µmol/kg) and fetal serum (0 -0.66 µmol/L , mean 0.23 µmol/L ), and fetal urine (4.1-174.1 µmol/L mean 38.5 µmol/L ), with fetal levels near biomolar equivalent to maternal levels (maternal serum 0.18-0.86 µmol/L , mean 0.46 µmol/L; maternal urine 42.6-254.0 µmol/L , mean 149.3 µmol/L). WSD feeding neither accelerated nor reduced metformin bioaccumulations in maternal or fetal serum, urine, amniotic fluid, placenta nor fetal tissues. In these 11 animals, fetal bioaccumulation of metformin was associated with less fetal skeletal muscle (57% lower cross-sectional area of gastrocnemius) and decreased liver, heart, and retroperitoneal fat masses (p<0.05), collectively driving lower delivery weight (p<0.0001) without changing the crown-rump length. Sagittal sections of fetal kidneys demonstrated delayed maturation, with disorganized glomerular generations and increased cortical thickness; this renal dysmorphology was not accompanied by structural nor functional changes indicative of renal insufficiency. CONCLUSIONS: We demonstrate fetal bioaccumulation of metformin with associated fetal growth restriction and renal dysmorphology following maternal initiation of the drug within 30 days of conception in primates. Given these results and the prevalence of metformin use during pregnancy, additional investigation of any potential immediate and enduring effects of prenatal metformin use is warranted.

Discrete placental gene expression signatures accompany diabetic disease classifications during pregnancy.

BACKGROUND: Gestational diabetes mellitus (GDM) affects up to 10% of pregnancies and is classified into subtypes GDMA1 (managed by lifestyle modifications) and GDMA2 (requiring medication). However, whether these subtypes are distinct clinical entities or more reflective of an extended spectrum of normal pregnancy endocrine physiology remains unclear. OBJECTIVE: Integrated bulk RNA-sequencing (RNA-seq), single-cell RNA-sequencing (scRNA-seq), and spatial transcriptomics harbors the potential to reveal disease gene signatures in subsets of cells and tissue microenvironments. We aimed to combine these high-resolution technologies with rigorous classification of diabetes subtypes in pregnancy. We hypothesized that differences between pre-existing Type 2 and gestational diabetes subtypes would be associated with altered gene expression profiles in specific placental cell populations. STUDY DESIGN: In a large case-cohort design, we compared validated cases of GDMA1, GDMA2, and type 2 diabetes (T2DM) to healthy controls by bulk RNA-seq (n=54). Quantitative analyses with RT-qPCR of presumptive genes of significant interest were undertaken in an independent and non-overlapping validation cohort of similarly well-characterized cases and controls (n=122). Additional integrated analyses of term placental single-cell, single-nuclei, and spatial transcriptomics data enabled us to determine the cellular subpopulations and niches that aligned with the GDMA1, GDMA2, and T2DM gene expression signatures at higher resolution and with greater confidence. RESULTS: Dimensional reduction of the bulk RNA-seq data revealed that the most common source of placental gene expression variation was the diabetic disease subtype. Relative to controls, we found 2,052 unique and significantly differentially expressed genes (-22 thresholds; q<0.05 Wald Test) among GDMA1 placental specimens, 267 among GDMA2, and 1,520 among T2DM. Several candidate marker genes (CSH1, PER1, PIK3CB, FOXO1, EGFR, IL2RB, SOD3, DOCK5, and SOGA1) were validated in an independent and non-overlapping validation cohort (q<0.05 Tukey). Functional enrichment revealed the pathways and genes most impacted for each diabetes subtype, and the degree of proximal similarity to other subclassifications. Surprisingly, GDMA1 and T2DM placental signatures were more alike by virtue of increased expression of chromatin remodeling and epigenetic regulation genes, while albumin was the top marker for GDMA2 with increased expression of placental genes in the wound healing pathway. Assessment of these gene signatures in single-cell, single-nuclei, and spatial transcriptomics data revealed high specificity and variability by placental cell and microarchitecture types. For example, at the cellular and spatial (e.g., microarchitectural) levels, distinguishing features were observed in extravillous trophoblasts (GDMA1) and macrophages (GDMA2). Lastly, we utilized these data to train and evaluate four machine learning models to estimate our confidence in predicting the control or diabetes status of placental transcriptome specimens with no available clinical metadata. CONCLUSION: Consistent with the distinct association of perinatal outcome risk, placentae from GDMA1, GDMA2, and T2DM-affected pregnancies harbor unique gene signatures that can be further distinguished by altered placental cellular subtypes and microarchitectural niches.

SARS-CoV-2 niches in human placenta revealed by spatial transcriptomics.

BACKGROUND: Functional placental niches are presumed to spatially separate maternal-fetal antigens and restrict the vertical transmission of pathogens. We hypothesized a high-resolution map of placental transcription could provide direct evidence for niche microenvironments with unique functions and transcription profiles. METHODS: We utilized Visium Spatial Transcriptomics paired with H&E staining to generate 17,927 spatial transcriptomes. By integrating these spatial transcriptomes with 273,944 placental single-cell and single-nuclei transcriptomes, we generated an atlas composed of at least 22 subpopulations in the maternal decidua, fetal chorionic villi, and chorioamniotic membranes. FINDINGS: Comparisons of placentae from uninfected healthy controls (n = 4) with COVID-19 asymptomatic (n = 4) and symptomatic (n = 5) infected participants demonstrated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in syncytiotrophoblasts occurred in both the presence and the absence of maternal clinical disease. With spatial transcriptomics, we found that the limit of detection for SARS-CoV-2 was 1/7,000 cells, and placental niches without detectable viral transcripts were unperturbed. In contrast, niches with high SARS-CoV-2 transcript levels were associated with significant upregulation in pro-inflammatory cytokines and interferon-stimulated genes, altered metallopeptidase signaling (TIMP1), with coordinated shifts in macrophage polarization, histiocytic intervillositis, and perivillous fibrin deposition. Fetal sex differences in gene expression responses to SARS-CoV-2 were limited, with confirmed mapping limited to the maternal decidua in males. CONCLUSIONS: High-resolution placental transcriptomics with spatial resolution revealed dynamic responses to SARS-CoV-2 in coordinate microenvironments in the absence and presence of clinically evident disease. FUNDING: This work was supported by the NIH (R01HD091731 and T32-HD098069), NSF (2208903), the Burroughs Welcome Fund and the March of Dimes Preterm Birth Research Initiatives, and a Career Development Award from the American Society of Gene and Cell Therapy.

Identifying the Transcriptional Response of Cancer and Inflammation-Related Genes in Lung Cells in Relation to Ambient Air Chemical Mixtures in Houston, Texas.

Atmospheric pollution represents a complex mixture of air chemicals that continually interact and transform, making it difficult to accurately evaluate associated toxicity responses representative of real-world exposure. This study leveraged data from a previously published article and reevaluated lung cell transcriptional response induced by outdoor atmospheric pollution mixtures using field-based exposure conditions in the industrialized Houston Ship Channel. The tested hypothesis was that individual and co-occurring chemicals in the atmosphere relate to altered expression of critical genes involved in inflammation and cancer-related processes in lung cells. Human lung cells were exposed at an air-liquid interface to ambient air mixtures for 4 h, with experiments replicated across 5 days. Real-time monitoring of primary and secondary gas-phase pollutants, as well as other atmospheric conditions, was simultaneously conducted. Transcriptional analysis of exposed cells identified critical genes showing differential expression associated with both individual and chemical mixtures. The individual pollutant identified with the largest amount of associated transcriptional response was benzene. Tumor necrosis factor (TNF) and interferon regulatory factor 1 (IRFN1) were identified as key upstream transcription factor regulators of the cellular response to benzene. This study is among the first to measure lung cell transcriptional responses in relation to real-world, gas-phase air mixtures.

Conditional postnatal deletion of the neonatal murine hepatic circadian gene, Npas2, alters the gut microbiome following restricted feeding.

BACKGROUND: We have recently shown in both non-human primates and in rodents that fetal and neonatal hepatic expression of the circadian transcription factor, Npas2, is modulated by a high fat maternal diet and plays a critical role in establishing life-long metabolic homeostasis. Similarly, we and others have also established the importance of the maternal and early postnatal diet on establishment of the early gut microbiome. OBJECTIVE: We hypothesized that altered circadian gene expression solely in the neonatal liver would result in gut microbiome dysbiosis, especially with diet-induced metabolic stress (ie, restricted feeding). Using a murine model in which we conditionally knock out Npas2 in the neonatal liver, we aimed to determine the role of the circadian machinery in gut dysbiosis with restricted feeding. STUDY DESIGN: We collected fecal samples from liver Npas2 conditional knockout (n = 11) and wild-type (n = 13) reproductive-aged mice before (study day 0) and after the restricted feeding study (study day 17). Extracted DNA was sequenced using the MiSeq Illumina platform using primers specific for the V4 region of the 16S ribosomal DNA gene. The resulting sequences were quality filtered, aligned, and assigned taxonomy. Principal coordinate analysis was performed on unweighted and weighted UniFrac distances between samples with a permutation analysis of variance to assess clustering significance between groups. Microbial taxa that significantly differ between groups of interest was determined using linear discriminate analysis effect size and randomForrest. RESULTS: Principal coordinate analysis performed on weighted UniFrac distances between male conditional knockout and wild-type cohorts revealed that the gut microbiome of the mice did not differ by genotype at the start of the restricted feeding study but did differ by virtue of genotype at the end of the study (P = .001). Moreover, these differences could be at least partially attributed to restricted feeding-associated alterations in relative abundance of the Bacteroides genus, which has been implicated as crucial to establishing a healthy gut microbiome early in development. CONCLUSION: Here we have provided an initial key insight into the interplay between neonatal establishment of the peripheral circadian clock in the liver and the ability of the gut microbiome to respond to dietary and metabolic stress. Because Npas2 expression in the liver is a target of maternal high-fat diet-induced metabolic perturbations during fetal development, we speculate that these findings have potential implications in the long-term metabolic health of their offspring.

Structural and transcriptomic response to antenatal corticosteroids in an Erk3-null mouse model of respiratory distress.

BACKGROUND: Neonatal respiratory distress syndrome in preterm infants is a leading cause of neonatal death. Pulmonary insufficiency-related infant mortality rates have improved with antenatal glucocorticoid treatment and neonatal surfactant replacement. However, the mechanism of glucocorticoid-promoted fetal lung maturation is not understood fully, despite decades of clinical use. We previously have shown that genetic deletion of Erk3 in mice results in growth restriction, cyanosis, and early neonatal lethality because of pulmonary immaturity and respiratory distress. Recently, we demonstrated that the addition of postnatal surfactant administration to antenatal dexamethasone treatment resulted in enhanced survival of neonatal Erk3-null mice. OBJECTIVE: To better understand the molecular underpinnings of corticosteroid-mediated lung maturation, we used high-throughput transcriptomic and high-resolution morphologic analysis of the murine fetal lung. We sought to examine the alterations in fetal lung structure and function that are associated with neonatal respiratory distress and antenatal glucocorticoid treatment. STUDY DESIGN: Dexamethasone (0.4 mg/kg) or saline solution was administered to pregnant dams on embryonic days 16.5 and 17.5. Fetal lungs were collected and analyzed by microCT and RNA-seq for differential gene expression and pathway interactions with genotype and treatment. Results from transcriptomic analysis guided further investigation of candidate genes with the use of immunostaining in murine and human fetal lung tissue. RESULTS: Erk3(-/-) mice exhibited atelectasis with decreased overall porosity and saccular space relative to wild type, which was ameliorated by glucocorticoid treatment. Of 596 differentially expressed genes (q < 0.05) that were detected by RNA-seq, pathway analysis revealed 36 genes (q < 0.05) interacting with dexamethasone, several with roles in lung development, which included corticotropin-releasing hormone and surfactant protein B. Corticotropin-releasing hormone protein was detected in wild-type and Erk3(-/-) lungs at E14.5, with significantly temporally altered expression through embryonic day 18.5. Antenatal dexamethasone attenuated corticotropin-releasing hormone at embryonic day 18.5 in both wild-type and Erk3(-/-) lungs (0.56-fold and 0.67-fold; P < .001). Wild type mice responded to glucocorticoid administration with increased pulmonary surfactant protein B (P = .003). In contrast, dexamethasone treatment in Erk3(-/-) mice resulted in decreased surfactant protein B (P = .012). In human validation studies, we confirmed that corticotropin-releasing hormone protein is present in the fetal lung at 18 weeks of gestation and increases in expression with progression towards viability (22 weeks of gestation; P < .01). CONCLUSION: Characterization of whole transcriptome gene expression revealed glucocorticoid-mediated regulation of corticotropin-releasing hormone and surfactant protein B via Erk3-independent and -dependent mechanisms, respectively. We demonstrated for the first time the expression and temporal regulation of corticotropin-releasing hormone protein in midtrimester human fetal lung. This unique model allows the effects of corticosteroids on fetal pulmonary morphologic condition to be distinguished from functional gene pathway regulation. These findings implicate Erk3 as a potentially important molecular mediator of antenatal glucocorticoid action in promoting surfactant protein production in the preterm neonatal lung and expanding our understanding of key mechanisms of clinical therapy to improve neonatal survival.

From the Field to the Laboratory: Air Pollutant-Induced Genomic Effects in Lung Cells.

Current in vitro studies do not typically assess cellular impacts in relation to real-world atmospheric mixtures of gases. In this study, we set out to examine the feasibility of measuring biological responses at the level of gene expression in human lung cells upon direct exposures to air in the field. This study describes the successful deployment of lung cells in the heavily industrialized Houston Ship Channel. By examining messenger RNA (mRNA) levels from exposed lung cells, we identified changes in genes that play a role as inflammatory responders in the cell. The results show anticipated responses from negative and positive controls, confirming the integrity of the experimental protocol and the successful deployment of the in vitro instrument. Furthermore, exposures to ambient conditions displayed robust changes in gene expression. These results demonstrate a methodology that can produce gas-phase toxicity data in the field.

Administration of antenatal glucocorticoids and postnatal surfactant ameliorates respiratory distress syndrome-associated neonatal lethality in Erk3(-/-) mouse pups.

BACKGROUND: Respiratory distress syndrome (RDS) persists as a prevalent cause of infant morbidity and mortality. We have previously demonstrated that deletion of Erk3 results in pulmonary immaturity and neonatal lethality. Using RNA sequencing, we identified corticotrophin releasing hormone (CRH) and surfactant protein B (SFTPB) as potential molecular mediators of Erk3-dependent lung maturation. In this study, we characterized the impact of antenatal glucocorticoids and postnatal surfactant on neonatal survival of Erk3 null mice. METHODS: In a double crossover design, we administered dexamethasone (dex) or saline to pregnant dams during the saccular stage of lung development, followed by postnatal surfactant or saline via inhalation intubation. Survival was recorded, and detailed lung histological analysis and staining for CRH and SFTPB protein expression were performed. RESULTS: Without treatment, Erk3 null pups die within 6 h of birth with reduced aerated space, impaired thinning of the alveolar septa, and abundant glycogen stores, as described in human RDS. The administration of dex and surfactant improved RDS-associated lethality of Erk3(-/-) pups and partially restored functional fetal lung maturation by accelerating the downregulation of pulmonary CRH and partially rescuing the production of SFTPB. CONCLUSION: These findings emphasize that Erk3 is integral to terminal differentiation of type II cells, SFTPB production, and fetal pulmonary maturity.

Transcriptome profiling of microRNA by Next-Gen deep sequencing reveals known and novel miRNA species in the lipid fraction of human breast milk.

While breast milk has unique health advantages for infants, the mechanisms by which it regulates the physiology of newborns are incompletely understood. miRNAs have been described as functioning transcellularly, and have been previously isolated in cell-free and exosomal form from bodily liquids (serum, saliva, urine) and tissues, including mammary tissue. We hypothesized that breast milk in general, and milk fat globules in particular, contain significant numbers of known and limited novel miRNA species detectable with massively parallel sequencing. Extracted RNA from lactating mothers before and following short-term treatment with recombinant human growth hormone (rhGH) was smRNA-enriched. smRNA-Seq was performed to generate 124,110,646 36-nt reads. Of these, 31,102,927 (25%) exactly matched known human miRNAs; with relaxing of stringency, 74,716,151 (60%) matched known miRNAs including 308 of the 1018 (29%) mature miRNAs (miRBase 16.0). These miRNAs are predicted to target 9074 genes; the 10 most abundant of these predicted to target 2691 genes with enrichment for transcriptional regulation of metabolic and immune responses. We identified 21 putative novel miRNAs, of which 12 were confirmed in a large validation set that included cohorts of lactating women consuming enriched diets. Of particular interest, we observed that expression of several novel miRNAs were altered by the perturbed maternal diet, notably following a high-fat intake (p<0.05). Our findings suggest that known and novel miRNAs are enriched in breast milk fat globules, and expression of several novel miRNA species is regulated by maternal diet. Based on robust pathway mapping, our data supports the notion that these maternally secreted miRNAs (stable in the milk fat globules) play a regulatory role in the infant and account in part for the health benefits of breast milk. We further speculate that regulation of these miRNA by a high fat maternal diet enables modulation of fetal metabolism to accommodate significant dietary challenges.

Maternal high-fat diet modulates the fetal thyroid axis and thyroid gene expression in a nonhuman primate model.

Thyroid hormone (TH) is an essential regulator of both fetal development and energy homeostasis. Although the association between subclinical hypothyroidism and obesity has been well studied, a causal relationship has yet to be established. Using our well-characterized nonhuman primate model of excess nutrition, we sought to investigate whether maternal high-fat diet (HFD)-induced changes in TH homeostasis may underlie later in life development of metabolic disorders and obesity. Here, we show that in utero exposure to a maternal HFD is associated with alterations of the fetal thyroid axis. At the beginning of the third trimester, fetal free T(4) levels are significantly decreased with HFD exposure compared with those of control diet-exposed offspring. Furthermore, transcription of the deiodinase, iodothyronine (DIO) genes, which help maintain thyroid homeostasis, are significantly (P < 0.05) disrupted in the fetal liver, thyroid, and hypothalamus. Genes involved in TH production are decreased (TRH, TSHR, TG, TPO, and SLC5A5) in hypothalamus and thyroid gland. In experiments designed to investigate the molecular underpinnings of these observations, we observe that the TH nuclear receptors and their downstream regulators are disrupted with maternal HFD exposure. In fetal liver, the expression of TH receptor ß (THRB) is increased 1.9-fold (P = 0.012). Thorough analysis of the THRB promoter reveals a maternal diet-induced alteration in the fetal THRB histone code, alongside differential promoter occupancy of corepressors and coactivators. We speculate that maternal HFD exposure in utero may set the stage for later in life obesity through epigenomic modifications to the histone code, which modulates the fetal thyroid axis.

Maternal tobacco use modestly alters correlated epigenome-wide placental DNA methylation and gene expression.

Several studies linking alterations in differential placental methylation with pregnancy disorders have implicated (de)regulation of the placental epigenome with fetal programming and later-in-life disease. We have previously demonstrated that maternal tobacco use is associated with alterations in promoter methylation of placental CYP1A1 and that these changes are correlated with CYP1A1 gene expression and fetal growth restriction. In this study we sought to expand our analysis of promoter methylation by correlating it to gene expression on a genome-wide scale. Employing side-by-side IlluminaHG-12 gene transcription with Infinium27K methylation arrays, we interrogated correlative changes in placental gene expression and DNA methylation associated with maternal tobacco smoke exposure at an epigenome-wide level and in consideration of signature gene pathways. We observed that the expression of 623 genes and the methylation of 1024 CpG dinucleotides are significantly altered among smokers, with only 38 CpGs showing significant differential methylation (differing by a methylation level of ≥10%). We identified a significant Pearson correlation (≥0.7 or ≤-0.7) between placental transcriptional regulation and differential CpG methylation in only 25 genes among non-smokers but in 438 genes among smokers (18-fold increase, p < 0.0001), with a dominant effect among oxidative stress pathways. Differential methylation at as few as 6 sites was attributed to maternal smoking-mediated birth weight reduction in linear regression models with Bonferroni correction (p < 1.8 × 10(-6)). These studies suggest that a common perinatal exposure (such as maternal smoking) deregulates placental methylation in a CpG site-specific manner that correlates with meaningful alterations in gene expression along signature pathways.

Maternal tobacco use is associated with increased markers of oxidative stress in the placenta.

OBJECTIVE: We sought to extend our prior observations and histopathologically characterize key metabolic enzymes (CYP1A1) with markers of oxidative damage in the placental sections from smokers. STUDY DESIGN: Placental specimens were collected from term singleton deliveries from smokers (n = 10) and nonsmokers (n = 10) and subjected to a detailed histopathological examination. To quantify the extent of oxidative damage, masked score-graded (0-6) histopathology against 4-hydroxy-2-nonenal (4-HNE) and 8-hydroxydeoxyguanisine (8-OHdG) was performed. Minimal significance (P < .05) was determined with a Fisher's exact and a 2-tailed Student t test as appropriate. RESULTS: We observed a significant increase in the presence of syncytial knots in placentas from smokers (70% vs 10%, P = .02). These gross observations were accompanied by a significant aberrant placental aromatic hydrocarbon metabolism (increased CYP1A1, 4.4 vs 2.1, P = .002) in addition to evidence of oxidative damage (4-HNE 3.4 vs 1.1, P = .00005; 8-OHdG 4.9 vs 3.1, P = .0038). CONCLUSION: We observed a strong association between maternal tobacco use and aberrant placental metabolism, syncytial knot formation, and multiple markers of oxidative damage.

Epigenomics: maternal high-fat diet exposure in utero disrupts peripheral circadian gene expression in nonhuman primates.

The effect of in utero exposure to a maternal high-fat diet on the peripheral circadian system of the fetus is unknown. Using mRNA copy number analysis, we report that the components of the peripheral circadian machinery are transcribed in the nonhuman primate fetal liver in an intact phase-antiphase fashion and that Npas2, a paralog of the Clock transcription factor, serves as the rate-limiting transcript by virtue of its relative low abundance (10- to 1000-fold lower). We show that exposure to a maternal high-fat diet in utero significantly alters the expression of fetal hepatic Npas2 (up to 7.1-fold, P<0.001) compared with that in control diet-exposed animals and is reversible in fetal offspring from obese dams reversed to a control diet (1.3-fold, P>0.05). Although the Npas2 promoter remains largely unmethylated, differential Npas2 promoter occupancy of acetylation of fetal histone H3 at lysine 14 (H3K14ac) occurs in response to maternal high-fat diet exposure compared with control diet-exposed animals. Furthermore, we find that disruption of Npas2 is consistent with high-fat diet exposure in juvenile animals, regardless of in utero diet exposure. In summary, the data suggest that peripheral Npas2 expression is uniquely vulnerable to diet exposure.

In utero tobacco exposure epigenetically modifies placental CYP1A1 expression.

The metabolic pathways used by higher-eukaryotic organisms to deal with potentially carcinogenic xenobiotic compounds from tobacco smoke have been well characterized. Carcinogenic compounds such as polycyclic aromatic hydrocarbons are metabolized sequentially in 2 phases: in phase I, CYP1A1 catalyzes conversion into harmful hydrophilic DNA adducts, whereas in phase II, GSTT1 enables excretion via conjugation into polar electrophiles. In an effort to understand susceptibility to in utero tobacco exposure, we previously characterized known metabolic functional polymorphisms and demonstrated that although deletion of fetal GSTT1 significantly modified birth weight in smokers, no polymorphism fully accounted for fetal growth restriction. Because smoking up-regulates CYP1A1 expression, we hypothesized that nonallelic (epigenetic) dysregulation of placental CYP1A1 expression via alterations in DNA methylation (meCpG) may further modify fetal growth. In the present article, we compared placental expression of multiple CYP family members among gravidae and observed significantly increased CYP1A1 expression among smokers relative to controls (4.4-fold, P < .05). To fully characterize CYP1A1 meCpG status, bisulfite modification and sequencing of the entire proximal 1-kilobase promoter (containing 59 CpG sites) were performed. CpG sites immediately proximal to the 5'-xenobiotic response element transcription factor binding element were significantly hypomethylated among smokers (55.6% vs 45.9% meCpG, P = .027), a finding that uniquely correlated with placental gene expression (r = 0.737, P = .007). Thus, in utero tobacco exposure significantly increases placental CYP1A1 expression in association with differential methylation at a critical xenobiotic response element.

Dyslipidemia and auditory function.

The relationship between dyslipidemia and hearing is unclear. This study was conducted to investigate whether elevated serum lipid levels impact auditory function in humans and in guinea pigs. In the human study, a cross-sectional study of 40 volunteers with dyslipidemia was conducted. Pure tone thresholds, distortion product otoacoustic emissions, and lipid profiles were analyzed. When controlled for patient age and sex, we found that elevated triglycerides were associated with reduced hearing. In the guinea pig study, a prospective study of animals fed a high-fat diet for 14 weeks was conducted. Although the high-fat diet led to a dramatic elevation in the average weight and total cholesterol in all animals (from 61 to 589 mg/dl), there were no meaningful changes in distortion product otoacoustic emission magnitudes. These results suggest that whereas chronic dyslipidemia associated with elevated triglycerides may reduce auditory function, short-term dietary changes may not.

Urocortin-deficient mice display normal stress-induced anxiety behavior and autonomic control but an impaired acoustic startle response.

Corticotropin-releasing hormone (Crh) plays an important role in modulating physiological and behavioral responses to stress. Its actions are mediated through two receptors, Crhr1 and Crhr2. Urocortin (Ucn), a Crh-related neuropeptide and the postulated endogenous ligand for Crhr2, is a potential mediator of stress responses. We generated Ucn-deficient mice using embryonic stem cell technology to determine its role in stress-induced behavioral and autonomic responses. Unlike Crhr1- or Crhr2-deficient mice, Ucn-deficient mice exhibit normal anxiety-like behavior as well as autonomic regulation in response to stress. However, the mutant mice display an impaired acoustic startle response that is not due to an obvious hearing defect. Thus, our results suggest that Ucn does not play an essential role in stress-induced behavioral and autonomic responses. Ucn may modulate the acoustic startle response through the Ucn-expressing neuron projections from the region of the Edinger-Westphal nucleus.