16th Annual Pioneers in Endocrinology Workshop Program

“Everything We Think We Know About Placental Extracellular Vesicles May Be Wrong: NanoFACS Is a Solution”

Terry K. Morgan, M.D., Ph.D.

Professor of Pathology, Obstetrics & Gynecology, and Biomedical Engineering
Oregon Health & Science University

“Sweet Beginnings and Sour Ends: Role of Maternal Diet, Diabetes, and Metformin on Fetal Programming”

Kjersti Aagaard, M.D., Ph.D., F.A.C.O.G.

National Director of Perinatal Research and Medical Director of Maternal Fetal Medicine of Texas HCA Healthcare
Research Scientist
Maternal Fetal Care Center
Division of Fetal Medicine and Surgery
Boston Children’s Hospital
Harvard Medical School

Abstracts

 

Maternal Fetal Unit (MFU)

MFU1. Placental Barrier Transport: Identification of Maternal and Infant Determinants of BCRP and MDR1 Transporter Levels
L. Aleksunes
Rutgers University

Objective: The ‘placenta barrier’ is comprised of membrane transporters including BCRP and MDR1 that actively efflux chemicals from the syncytiotrophoblast back to the maternal circulation. This function allows BCRP and MDR1 to limit the accumulation of drugs and toxicants and prevents transplacental transfer of substrates. In this study, we evaluated relationships between maternal (gestational weight gain, early pregnancy body mass index, smoking, parity, pregnancy complications, age) and infant factors (birth weight, race, transporter genetics) and placental levels of BCRP and MDR1 proteins in a healthy U.S. cohort. Methods: Term placentas were collected from healthy participants in the Understanding Pregnancy Signals and Infant Development Study (UPSIDE) birth cohort (Rochester, NY, n=237). BCRP and MDR1 proteins were quantified in frozen villous placenta tissues using quantitative targeted absolute proteomic mass spectrometry. We examined determinants of placental protein concentrations through bivariate analyses and multivariable linear regression models. Results: Levels of BCRP and MDR1 proteins in term placentas were moderately correlated (r=0.58, p<0.001). In bivariate analyses, BCRP levels were associated with gestational weight gain, infant race, parity, and the rs2231142 genetic variant. In mutually adjusted models, only parity (β=0.07; 95%CI:0.02,0.11) and the rs2231142 AC/AA genotype (β=-0.09, 95%CI:-0.14,-0.04) showed notable associations with BCRP levels. By comparison, in bivariate analyses, MDR1 levels were associated with parity and more weakly, with birthweight, race, smoking, and the rs1045642 and rs2032582 gene variants. In mutually adjusted models, parity (β=0.11; 95%CI:0.05,0.17), smoking (β=-0.11; 95%CI:-0.21,-0.01), the rs1046642 TT genotype (β=-0.14, 95%CI:-0.24,-0.03), and the rs2032582 TT genotype (β=0.10; 95%CI:-0.02,0.21) were associated with MDR1 levels. Conclusion: Identifying maternal and infant factors that contribute to the regulation of BCRP and MDR1 levels in healthy, term placentas is the first step in dissecting the impact of disease and environment on the integrity of the placental barrier. Supported by R01ES029275, R01HD083369, UG3OD023349, UH3OD023349, P30ES005022, UL1TR003017, and UC2HD113039.

MFU2. Prenatal Exposure to Cadmium alters Placental Morphology, Vasculature Development, and Macrophage Subpopulations in Mice
L. Aleksunes, B. Ames, C. Jiang, S. Wang, D. Kozlosky, S. Stratton, C. Gardner
Joint Graduate Program in Toxicology, Environmental and Occupational Health Sciences Institute, School of Public Health, Ernest Mario School of Pharmacy

Objective: The environmental metal cadmium (Cd) accumulates in the placenta during pregnancy and is associated with fetal growth restriction and preterm birth in rodents and humans. In the placenta, Cd generates oxidative stress which may be important in disrupting placenta development and macrophage responses. We sought to determine whether prenatal exposure of mice to CdCl2 alters placenta development and enrichment of macrophage populations. Methods: Pregnant C57BL/6Crl mice (n=9-10/group) received distilled water with CdCl2 (0, 5, or 50 ppm) ad libitum from gestational day (GD) 7-17. Tissues were collected on GD17 for ICP/MS quantification of Cd concentrations, histomorphologic assessment, and immunohistochemistry. Results: Exposure to CdCl2 did not affect fetal or placental weight or size, or number of resorptions. Histological analysis of the placentas demonstrated a significant decrease in junctional zone area, with no change in labyrinth zone size, in placentas of dams treated with 50 ppm CdCl2. Compared to vehicle-treated mice, the area of maternal and fetal blood vessels was decreased in placentas exposed to 5 and 50 ppm CdCl2. Immunohistochemical staining for the fetal endothelial surface marker CD34 was significantly increased in the placentas of fetuses exposed to 50 ppm CdCl2. Further, the number of macrophages that stained positive for the marker F4/80 was increased in both the labyrinth and junctional zones of placentas exposed to 50 ppm CdCl2. Conversely, the number of macrophages that stained positive for Iba1, a protein involved in phagocytosis, was decreased in the labyrinth zones of placentas exposed to 50 ppm CdCl2. Conclusion: In the absence of overt fetoplacental toxicity, exposure to CdCl2 during gestation altered development of placental zones and vasculature in mice as well as shifted the abundance of macrophage subpopulations. This research is supported by R01ES029275, T32ES007148, F31ES032319, P30ES005022, UC2HD113039, and Grover Fellowship.

MFU3. Transporter-Mediated Uptake of the Microcystin-LR Toxin in Human Placenta Cells at Different Oxygen Concentrations
L. Aleksunes, M. Campbell, M. Patel, Xia Wen, Shuo Xiao
Department of Pharmacology and Toxicology, Joint Graduate Program in Toxicology, Environmental and Occupational Health Sciences Institute, Rutgers, The State University of New Jersey

With global warming and waterway eutrophication, cyanobacterial harmful algal blooms are increasing in incidence and intensity, releasing toxins including microcystin-LR (MC-LR). While MC-LR is a well-established hepatotoxin and neurotoxin, there is growing interest in evaluating its female reproductive toxicities. We sought to evaluate the ability of MC-LR to enter and accumulate in trophoblasts at different oxygen concentrations. Intracellular accumulation of MC-LR (0.1, 1, and 10 µM) over 3 hrs was evaluated in immortalized human cytotrophoblasts (JAR and BeWo cells) and extravillous trophoblasts (HTR-8/SVneo) cells using western blotting. Moreover, the function of OATP transporters in JAR cells was determined by pre-incubating cells with cyclosporin A (10 µM), a general OATP inhibitor, prior to uptake of the OATP substrate fluorescein (1 µM) or MC-LR (1 µM) for up to 40 min. The impact of oxygen concentration on MC-LR uptake was tested by incubating JAR cells under 3, 8, and 20% O2 for 24 hrs, then exposing to MC-LR (1 µM) for 3 hrs. Concentration-dependent increases in MC-LR bound proteins (37 kDa) were observed in all three trophoblast cell lines. In addition, inhibition of OATP function in JAR cells using cyclosporin A caused a 57% reduction in MC-LR uptake. Compared to 20% O2, MC-LR uptake was decreased by 52% and 72% in JAR cells exposed to 3% and 8% O2 respectively. Microcystin-LR enters human trophoblasts by active OATP transporters in a concentration-dependent manner. This uptake is reduced at lower oxygen concentrations which are observed early in pregnancies and during pathological conditions. Ongoing research aims to identify specific OATP isoforms involved in MC-LR uptake into the placenta and the impact of microcystins on key placental functions.

MFU4. Differential Regulation of Placental OATP Transporters at Low Oxygen Concentrations
L. Aleksunes, C. Jiang, M. Campbell, X. Wen
Department of Pharmacology and Toxicology, Rutgers University Ernest Mario Department of Pharmacology and Toxicology, Rutgers University Ernest Mario School of Pharmacy, Piscataway, NJ 08854, USA. Environmental and Occupational Health Sciences Institute, Piscataway, NJ 08854, USA, School of Pharmacy, Piscataway, NJ 08854, USA, Joint Graduate Program in Toxicology, Rutgers University, Piscataway, NJ 08854, USA

The placenta is a dynamic tissue that begins as a hypoxic tissue with oxygen levels around 3% until blood flow increases and oxygen concentrations rise to 8% during the second and third trimesters. However, pregnancies with complications can decrease blood flow, resulting in reduced oxygen concentrations later in gestation. Organic anion transporting polypeptide (OATP) transporters are responsible for the cellular uptake of endobiotics and xenobiotics in the placenta. In this study, we sought to determine whether varying oxygen concentrations alter the mRNA expression of OATPs in a human immortalized trophoblast cell line. For this purpose, JAR choriocarcinoma cells were exposed to 20%, 8%, and 3% oxygen levels for 24 hours and OATP mRNAs were quantified by qPCR. To confirm that hypoxic responses were activated, downstream targets of hypoxia inducible factor-1α (HIF-1α) including glucose transporter 1 (GLUT1), heme oxygenase 1 (HO1), and vascular endothelial growth factor receptor 1 (VEGFR1) were quantified. Compared to atmospheric oxygen levels (20%), GLUT1 mRNAs were increased 4-fold and 8-fold in trophoblasts exposed to 8% and 3% oxygen, respectively. Similarly, VEGFR1 mRNA levels were up-regulated by 3-fold at 8% oxygen and HO-1 mRNA levels was increased by 2-fold at 3% oxygen. OATP mRNA levels were differentially regulated. Compared to 20% oxygen exposure, trophoblasts exposed to 3% oxygen exhibited a 50% decrease in OATP4A1 mRNAs. OATP1B3 decreased 1-fold from 8% to 3% oxygen. Conversely, OATP2B1 mRNAs increased 10- and 20-fold in trophoblasts exposed to 8% and 3% oxygen, respectively. OATP3A1 mRNAs were also up-regulated by 3-fold at 3% oxygen. These data suggest that OATPs are differentially regulated under low oxygen concentrations and may impact uptake of OATP substrates, including drugs and nutrients in the placenta.

 

Prenatal Exposure (PE)

PE1. Prenatal Exposure to Synthetic Chemicals in relation to HPA Axis Activity: A Systematic Review of the Epidemiological Literature
E. Barrett, A. Pande, C. Kinkade, N. Prout, S. Chowdhury, Z. Rivera-Núñez
Environmental and Occupational Health Sciences Institute, Rutgers University; Department of Biostatistics and Epidemiology, Rutgers School of Public Health, Robert Wood Johnson Medical School, Rutgers University; Wynne Center for Family Research, University of Rochester; Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry; Translational Biomedical Sciences Program, University of Rochester; Department of Biostatistics and Epidemiology, Rutgers School of Public Health.

Pregnant people are widely exposed to numerous synthetic chemicals with known endocrine-disrupting properties (e.g., phthalates, phenols, per- and poly-fluoroalkyl substances (PFAS)). To date, most epidemiological research on how endocrine-disrupting chemicals (EDCs) disrupt hormone pathways has focused on estrogens, androgens, and thyroid hormones. Far less research has examined the impact of EDCs on the hypothalamic-pituitary-adrenal (HPA) axis, despite its central role in the physiologic stress response and metabolic function. The objective of this study is to systematically review the epidemiological literature on prenatal synthetic EDC exposures in relation to HPA axis hormones (e.g., corticotropin-releasing hormone, adrenocorticotropic hormone, cortisol, cortisone) in pregnant people and their offspring. A literature search of PubMed, Scopus, and Embase was conducted. Primary research studies were selected for inclusion by two independent reviewers and risk of bias was assessed using the Office of Health Assessment and Translation guidelines established by the National Toxicology Program with customization for the specific research topic. Data were extracted from each study and included in a qualitative synthesis. A total of 22 published studies met the inclusion criteria. Phthalates were the most prevalent EDC studied, followed by PFAS, phenols, and parabens, with fewer studies considering other synthetic chemicals. Offspring glucocorticoids were the most commonly considered outcome, followed by maternal glucocorticoids and placental corticotropin-releasing hormone. There was considerable heterogeneity in methods across studies, particularly in HPA axis outcome measures and matrices, making cross-study comparisons challenging. Numerous studies suggested disruption of HPA axis hormones and sex differences in association, but results varied considerably across studies and EDC classes. The limited literature to date suggests the HPA axis may be vulnerable to disruption by synthetic EDCs. Carefully designed studies that prioritize biospecimen collection specific to HPA axis hormones are needed along with greater standardization of biospecimen collection and analysis protocols to facilitate cross-study comparisons and interpretation.

PE2. Prenatal mycoestrogen exposure in relation to infant cognitive and language development
E. Barrett, G. Lee, C. Kinkade, Z. Rivera-Núñez, M. Hansel, L. Aleksunes, T. O’Connor
Rutgers University – New Brunswick, Rutgers School of Public Health, University of Rochester

Zearalenone (ZEN), an estrogenic fungal-derived mycotoxin commonly found in cereal grains and processed foods, is linked to adverse effects on female reproduction in animal models. As climate change leads to warmer and moist conditions promoting fungal growth, studying these mycotoxins has become a priority. Given that prenatal estrogens (and environmental estrogens) have also been linked to neurodevelopmental outcomes in children, we now examine (for the first time) prenatal mycoestrogen exposure in relation to infant cognitive and language development. In a community sample, UPSIDE (Rochester, NY), we measured ZEN in urine samples collected in each trimester. When the resulting infants were 6, 12, and 24 months old, trained study staff administered two subscales of the Bayley Scales of Infant and Toddler Development III, cognition and language (n=118). For both subscales, higher scores indicate more advanced development. We fitted multivariable linear regression models examining associations between log-transformed ZEN concentrations and Bayley scores. Given growing evidence that ZEN has sex-specific impacts on development, our primary models were stratified by child sex. ZEN was detectable in >93% of maternal urine samples at median concentration 0.15 ng/ml. Among female infants, prenatal ZEN concentrations tended to be inversely associated with Bayley scores at all outcome timepoints. Results were strongest in relation to first trimester ZEN concentrations. For example, a log-unit increase in first trimester urinary ZEN concentrations was associated with 4.24 unit (95%CI: -7.80, -0.68), 3.01 unit (95%CI: -6.65, 0.54), and 6.17 unit (95%CI: -10.19,-2.16) lower cognitive scores in female infants at 6, 12, and 24 months of age, respectively. In contrast, in males, results were largely null. This is the first study to examine prenatal exposure to mycoestrogens in relation to neurodevelopment in humans. Our results suggest potential sex differences that warrant replication in larger samples with more extended childhood follow-up, especially in females.

PE3. The impact of mycoestrogen exposure on CRH during pregnancy
E. Barrett, A. Juenke, C. Kinkade, Z. Rivera-Nunez
Rutgers Schools of Public Health; EOHSI, Rutgers School of Pharmacy, Department of Pharmacology and Toxicology

Zearalenone (ZEN), a highly prevalent mycotoxin, widely contaminates human food supplies and has been detected in >90% of pregnant people and children in U.S. cohort studies. A growing body of research has established its endocrine disrupting properties, with animal models showing altered hormone signaling and poor pregnancy outcomes in response to ZEN exposure. Among the hormones of greatest importance during gestation is placental corticotropin-releasing hormone (pCRH), which has been suggested as “placental clock” that regulates the timing of parturition. Here, utilizing data from a medically normal risk pregnancy cohort (UPSIDE; Rochester, NY; n=273), we investigated gestational exposure to ZEN and its metabolites in relation to pCRH across pregnancy. Based on samples collected in each trimester, ZEN was measured in urine using LC-MS, and pCRH was measured in serum using radioimmunoassay. We fitted minimally and fully adjusted trimester-specific linear regression models examining log-transformed pCRH in relation to three specific gravity-adjusted and log-transformed mycoestrogen measures: (1) ZEN; (2) its metabolite, α-zearalenol (AZOL); and (3) the sum of ZEN and its metabolites (∑mycoestrogens). Mycoestrogens were detected in 94, 91, and 90% of urine samples in the first, second, and third trimesters, respectively. Although no associations were observed in early to mid-pregnancy, in the third trimester, in fully adjusted models, mycoestrogens were positively associated with pCRH concentrations: ZEN (β: 0.06; 95%CI: -0.02, 0.14); aZOL (β: 0.08, 95%CI: 0.002, 0.16); ∑mycoestrogens (β: 0.06, 95%CI: -0.02, 0.14). Our results suggest mycoestrogen exposure may be associated with elevated pCRH in late pregnancy, a risk factor for preterm birth. Further modelling to examine changes in pCRH across pregnancy is underway as are analyses examining sex-specific associations.

PE4. Retinoic acid regulates pregnancy-induced heart remodeling
L. Quadro, Y. Kim, C. Holloway, N. Isoherranen, E. Madonna, U. Hammerling
Rutgers University, NIH, University of Washington

The maternal heart enlarges to adapt to prolonged increased volume overload. This physiological hypertrophy, associated with normal or increased ventricular function, is completely reversible post-partum. Failure of the heart to adapt during pregnancy could promote pathological conditions in the mother and/or diminish the ability of the maternal circulation to meet the demands of the growing fetus. The factors predisposing to maternal heart conditions still need to be fully elucidated. Vitamin A is emerging as a mediator of cardiometabolic health. This essential nutrient regulates both organs’ development and their functions in adulthood, mainly owing to the transcriptional role of its active metabolite, all-trans retinoic acid (RA). RA binds to specific nuclear receptors (RAR and RXR) to modulate cellular proliferation, differentiation, and metabolism. In mammalian tissue, optimal RA concentrations are maintained by balancing the endogenous synthesis of RA and its degradation. RA signaling has been implicated as cardioprotective against maladaptive heart remodeling as it prevented pathological cardiac hypertrophy in animal models and humans. However, nothing is known about the metabolism of vitamin A in the heart of the pregnant mother. Our data show that cardiac RA levels decline during pregnancy in wild-type (WT) mouse dams to normalize post-partum; expression of Cyp26B1 – the main enzyme that catabolizes RA in extrahepatic tissues – is also dramatically reduced in the heart of pregnant WT dams; the heart of pregnant WT dams treated with RA or its precursor retinaldehyde during gestation fails to enlarge at late-pregnancy; and the heart of nulliparous WT female mice enlarges upon pharmacological inhibition of RA signaling. Moreover, our data suggest the renin-angiotensin system (RAS) as one of the pathways whereby RA regulates maternal cardiac growth. Based on these findings, we hypothesized that RA is a critical regulator of pregnancy-induced cardiac adaptive remodeling.

 

Sex Differences (SD)

SD1. Effects of Chronic Stress on Avoidance Behavior in the Estrogen Receptor Alpha Knockout (ERKO) Mouse
T. Roepke, V. Appel, A. Yasrebi, T. Degroat, K. Wiersielis, B. Samuels
Department of Animal Sciences, School of Environmental and Biological Sciences, and Graduate Program in Endocrinology and Animal Biosciences, Rutgers University, New Brunswick, NJ 08901; Department of Psychology, School of Arts and Sciences, Rutgers University, Piscataway, NJ 08854

Chronic stress has long been implicated in the development of mood disorders. Research has demonstrated that cisgender women are more susceptible than cisgender men to developing such conditions, and that these sex differences may, in part, be attributed to endocrine pathways. The mechanisms behind these pathways are progressively being elucidated, with focus on estrogen signaling. Using estrogen receptor alpha knockout (ERKO) mice, we examined the role of chronic stress on avoidance behavior across the sexes. Experimental mice underwent six weeks of chronic variable mild stress (CVMS) to induce a depressive phenotype, while control mice were not stressed. Four behavior tests were used to assess avoidance behavior: the open field test, elevated plus maze, light-dark test, and novelty suppressed feeding. We demonstrated effects of sex and stress on behavior where chronic stress has an anxiogenic effect in both sexes, that was more prominent in females. When compared to our published work in wild-type mice, our findings suggest that the deletion of estrogen receptor alpha increases the susceptibility to chronic stress, which may also involve estrogen receptor beta or G-protein estrogen receptor 1 to elicit the observed avoidant responses. This work lends itself to further investigation into the intersection of chronic stress and steroid hormone signaling pathways.

SD2. Network Meta-analysis of Estrogen’s Effects on Signaling Pathways controlling RUNX-2 through regulation of novel ERG-PDLIM3 gene.
S. Chang, A. Hassan
Seton Hall University

Estrogens, including estrone (E1), estradiol (E2), estriol (E3), and estetrol (E4), regulate the female life cycle and play a crucial role in development, metabolism, and cell function. Throughout evolution, estrogen has regulated reproduction by affecting reproductive organ development and behavior. Estrogen impacts all vertebrates, including fish, and has a role in physiological and pathological states in both genders. RUNX-2 gene is a member of the RUNX family of transcription factors and encodes a nuclear protein with a Runt DNA-binding domain. This protein is essential for osteoblastic differentiation and skeletal morphogenesis and acts as a scaffold for nucleic acids and regulatory factors involved in skeletal gene expression. The protein can bind DNA both as a monomer or, with more affinity, as a subunit of a heterodimeric complex. In 2022, a study was conducted to characterize novel genes that are regulated by estrogen binding to its receptors (α or β). The PDLIM3 gene, with a coefficient of variation of 0.083, received the highest score among other genes. We found a strong correlation between estrogen binding to its receptors α or β, followed by expression of PDLIM3 gene, and activation of RUNX-2 expression through regulation of specifically miR-9 and miR-10. We also determined a novel miRNA that is integrated in the activation of RUNX-2 through transcription of PDLIM3 gene.

SD3. Sexual Dimorphism of Retinoid Metabolism in the Adult Lung
I. Shmarakov, O. Groh, O. Ilnytska, A. Yasrebi, V. Appel, T. Roepke
Rutgers University

The intrinsic sex variability in the physiology of the respiratory system governs the morbidity and mortality of lung diseases in humans and animals across the lifespan. Sex chromosomes and gonadal steroid hormones affect the differences in disease severity and lethality among men, women, and transgender/non-binary individuals presenting with acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS). However, the precise mechanisms underlying the differences in ALI/ARDS pathophysiology across the sexes remain to be understood. Retinoids (vitamin A and its metabolites) are essential factors required for maintaining a healthy alveolar microenvironment that act critically to buffer the lung against the adverse consequences of ALI/ARDS. We have identified a sex-associated variation in lung retinoid metabolism, with postpubertal females having significantly higher tissue retinoid concentrations as compared to the diet- and age-matched males. In addition, lung retinoid concentrations are highly responsive to estrogen signaling. Altered estrogen signaling in estrogen receptor knockout mice (Esr1-/-) leads to a decline in lung tissue retinoids in Esr1-/- females compared to wild-type females (Esr1+/+) and the absence of differences between Esr1-/- females and Esr1-/- males. Moreover, pharmacological manipulations with estrogen signaling in mice with testes or orchiectomized mice treated with estrogen supplementation result in lung retinoid elevation and acquisition of a retinoid phenotype similar to intact wild-type females. Importantly, when wild-type animals from both sexes are induced ALI/ARDS, female mice have significantly higher lethality. However, the sex variability in the ALI/ARDS survival rates disappears when animals of both sexes lack lung retinoids (Lrat-/- mice), suggesting that different lung retinoid metabolism may contribute to the differences in ALI/ARDS lethality between males and females. Taken together, our data established that estrogen signaling is involved in regulating lung retinoid homeostasis, a critical factor in maintaining a healthy alveolar microenvironment, thus may affect pathophysiological responses during ALI/ARDS.

 

Food Intake (FI)

FI1. Time-restricted eating and calorie restriction affects bone in older women: randomized controlled trial
S. Shapses, B. McGuire, Y. Schlussel, S. Malin, A. Shah, L. Zhao
Department of Nutritional Sciences, Rutgers University; N.J. Institute of Food, Nutrition and Health (NJ-IFNH); Department of Medicine, Rutgers Robert Wood Johnson Medical School; Department of Kinesiology & Health, Rutgers University; Department of Biochemistry and Microbiology, Rutgers University

Purpose: Obesity is increasing worldwide, and calorie restriction (CR) is recommended to reduce weight. However, weight loss can negatively affect bone. Bone turnover and the gut microbiota display circadian and feeding rhythms, and disruptions in these rhythms can occur due to extended eating windows (≥12 hours). Time-restricted eating (TRE) manipulates the temporal pattern of food intake to restore synchronicity of circadian rhythms. Peripheral clocks are in multiple organs, including bone, yet studies using TRE have not examined bone as a primary outcome. We hypothesized that TRE would attenuate bone loss during CR.
Methods: Postmenopausal women (BMI ≥25kg/m2, 50-79 years old) were randomized to six months of CR with or without TRE. TRE+CR were instructed to self-select a ≤9-hour eating window that ended 4 hours before their usual bedtime. CR were asked to follow their usual eating window without skipping meals while having an evening snack. Both groups attended counseling sessions to reduce calorie intake. Serum and fecal samples were collected to assess bone turnover and the gut microbiota. Bone mineral density (BMD) and bone quality were assessed by dual-energy x-ray absorptiometry and peripheral quantitative computed tomography at 0 and 24 weeks.
Results: One hundred and sixty-eight women were screened, 49 were recruited, and 41 completed the trial. At baseline, the women (61±5 years, 71% white, 33±2kg/m2) had an eating window of 12.3±0.9 hours/day. During the intervention, CR (n=19) women consumed food over 12.6±0.8 hours/day while TRE+CR (n=22) reduced the eating window to 8.1±0.7 hours (p<0.001). Per-protocol analysis indicated weight loss did not differ between CR and TRE+CR (-5.1±5.6 vs -7.5±3.9kg). Hip BMD decreased (p<0.001) and lumbar spine showed a trend to decrease (p<0.08) over time without differences between groups. There was a greater decline in BMD at the 33% radius in the CR (-0.016±0.024g/cm2) compared to TRE+CR (0.009±0.027g/cm2) (p<0.001). Total tibial volumetric BMD loss was also greater during CR compared to TRE+CR (p<0.05). Changes in trabecular, cortical, and bone geometry parameters were not different between groups.
Conclusion: Weight loss and hip and spine BMD were similar between groups but TRE attenuated BMD loss at the 33% radius and tibia in older women with overweight or obesity. Examining mechanisms to explain these findings are currently a focus in the lab.

FI2. Kratom (Mitragyna speciosa) alkaloids effect on hemodynamic and pulmonary measures in normal weight and obese mice.
N. Bello, S. Fields, A. Elango, E. Bernstein, Q. Wu, J. Simon
Rutgers, The State University of NJ; Nutritional Sciences Graduate Program; SEBS, Department of Animal Sciences; New Use Agriculture and Natural Plant Products Program (NUANPP) and Department of Plant Biology, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901, USA

Kratom is derived from the leaves of Mitragyna speciosa, an evergreen tree indigenous to Southeast Asia. Typically, kratom is sold as a natural product for pain management and recreational purposes, but also has been marketed for obesity management. The primary purpose of the project was to examine whether chronic oral dosing of a standardized alkaloid enriched-kratom extract (KE) prevented weight gain in diet-induced obese mice. The secondary purpose was to determine whether acute human equivalent doses of KE and major alkaloid, mitragynine (MTG), differentially influence cardiopulmonary measures in normal weight or obese mice. Kratom powder was standardized to a KE containing 62.5 ± 1.2 mg/g of MTG. For obesity prevention, male C57BL/6J mice (8 weeks old; n=24) received daily oral doses of vehicle, 50 mg/kg KE or 150 mg/kg coincident to a high fat diet (HFD; 45% fat) switch. On days 26-30, mice were assessed for hemodynamic parameters using a non-invasive CODA® mouse rat tail-cuff system. For acute dosing, normal weight (n = 16) or diet-induced obese (>13 weeks on HFD; n=14) mice received an oral dose of vehicle, 290-500 mg/kg KE, or 18-31.3 MTG prior to hemodynamic or DSI® whole body plethysmography assessments. For obesity prevention, there were no dose-dependent differences in body weight gain, but there was a blood-pressure lowering effect with reductions in systolic [F(2, 19) = 5.3, p <0.05], diastolic [F(2, 19) = 3.8, p< 0.05], and mean blood pressures [F(2, 19) = 4.7, p< 0.05] with KE 50 mg/kg reducing BP compared with vehicle (p<0.05 for all). For acute dosing, there was a dose effect [F(2, 56)=4.4, p< 0.05] with the 31.3 mg/kg MTG dose in normal weight compared with the obese mice (p < 0.05). There were no differences in pulmonary measures between normal and obese measures. However, there were dose suppressive effects of KE and MTG on breaths per minute (BPM) and ventilation rate (MvB). Overall, our preliminary findings do not support the use of kratom alkaloid extract in diet-induced obesity prevention. Our findings do suggest a differential effect of kratom products on hemodynamic measures related to weight gain in mice.

 

Neuroinflammation (N)

N1. Prenatal alcohol exposure causes microglia to release exosomes with elevated MIP-1α that promotes death of stress-regulatory proopiomelanocortin neurons via glutamate excitotoxicity.
D. Sarkar, P. Tarale, S. Chaudhary
Department of Animal Sciences, Rutgers, State University of New Jersey, New Brunswick, New Jersey, USA

Microglia is known to play a central role in pathophysiology of fetal alcohol syndrome disorders (FASD). However, prenatal alcohol exposure (PAE) mediated alteration in chemokine level within microglia-derived exosomes and its mechanistic implication in FASD has not been investigated. In the present study, we determine the chemokine profile of ethanol-activated microglial exosomes and studied the molecular pathway that induce exosome mediated death of stress regulatory proopiomelanocortin (POMC) neurons. Our chemokine multiplex immunoassay and further ELISA determine that ethanol (50mM) significantly elevates macrophage inflammatory protein (MIP)-1α/CCL3 within exosomes derived from rat’s hypothalamic microglia in culture. We further validated the elevated level of MIP-1α in microglial exosomes isolated from hypothalamic tissues collected from postnatal rats (both males and females) fed daily (PND2-PND6) with 2.5 mg/kg ethanol or control milk formula. These ethanol-activated microglial exosomes when added to β-endorphin/POMC neurons in culture causes MIP-1α-CCR5 signaling mediated overexpression of glutamate transporter-1 and NMDA receptor subunit genes which leads to increase calcium influx mediated glutamatergic excitotoxicity and immune-excitotoxicity induced apoptotic death. Maraviroc, antagonist for CCR5 was able to prevent apoptosis of POMC neurons in culture when pretreated prior to adding ethanol activated exosomes. Maraviroc treatment in postnatal rats prior feeding with alcohol was also able to reduce ethanol-induced POMC neuronal death in the hypothalamus. Also, we determined that postnatal treatment with maraviroc suppress the corticosterone stress response and improve the anxiety-related behavior in alcohol-fed rats during adolescence. Our investigation suggests that PAE causes microglia to release exosomes with elevated level of MIP-1α/CCL3 that promotes MIP-1α-CCR5 signaling mediated glutamate excitotoxicity induced apoptotic death of POMC neurons and related hormonal and behavioral stress response.

N2. Selective Impairment of Sustained Attention by High-Dose Orexin Antagonism: Implications for Ox2R Involvement.
M. James, S. O’Connor, N. Krishnakumar, K. Deak, M. Paladino, B. Gruszka, J. Wiskerke
Department of Psychology, Rutgers University; Department of Psychiatry, Brain Health Institute Robert Wood Johnson Med. School, Rutgers University; Department of Psychology, Texas A&M University; Rutgers University Grad. Program In Neuroscience; Linköping Univ., Linkoping, Sweden.

Introduction: The hypothalamic/orexin system modulates a spectrum of physiological and psychological functions. Although previous work has implicated the orexin system in arousal and attention, its role in sustained vigilance has not been tested. Understanding this mechanism could provide insights into how orexin signaling can be modulated in disorders related to attention and arousal, such as ADHD or narcolepsy. Here, we tested how manipulating the orexin system affects performance on a rodent psychomotor vigilance task (rPVT), a translationally relevant measure of sustained attention. Methods: Male (n=9) and female (n=8) orexin cre+ rats were trained on the rPVT, which required them to make responses on a lever following presentation of a light cue that varied in onset time (3-10s after trial initiation); correct responses were rewarded with sucrose pellets and sessions ran for 30min. Following training, rats were injected with a cre-dependent viral construct containing the inhibitory hM4Di DREADD into the orexin cell field. Rats were subsequently injected with JHU37160 (J60; 0.1mg/kg) prior to subsequent testing on the rPVT. Performance was also assessed following injections of the orexin 1 receptor antagonist, SB-334867 (0, 3, 10, 30mg/kg; i.p.) and RTIOX-276 (0, 5, 10, 20mg/kg; i.p.), orexin 2 receptor antagonist, MK-1064 (0, 5, 10, 20mg/kg; oral), the dual orexin receptor antagonist suvorexant (0, 3, 10, 30mg/kg; oral) and a combination of SB-334867 (10mg/kg; i.p.) and MK-1064 (10, 20mg/kg; oral). All compounds were tested in a within-subjects design (counterbalanced). Results: Across all measures, there was no effect of sex and thus data from males and females were analyzed together. Two subpopulations of rats emerged: those with high (>65% accuracy) vs low (<65% accuracy) baseline performance on the rPVT (HP vs LP). Inhibition of orexin neurons impaired accuracy on rPVT only in high performers whereas pharmacological blockade of Ox1R, but not Ox2R, reduced accuracy. Conclusion: Inhibition of orexin neurons impairs measures of sustained attention on rPVT. Dual antagonism of Ox1R and Ox2R produced more pronounced impairments in performance, indicating a potential synergistic role for signaling at these receptors in maintaining sustained vigilance.

N3. Bisphenol-a exposure alters puberty onset and orexin (hypocretin) function in adulthood: a role for microglia.
M. James, M. Bilotti, C. Brown, N. Bello, T. Roepke
RBHS; Psychiatry, Joint Graduate Program in Toxicology, Rutgers Brain Health Institute, School of Environmental and Biological Sciences.

The timing of puberty is determined by a complex interplay between various endogenous factors, but it can also be significantly influenced by environmental cues. We previously showed that exposure to bisphenol A (BPA) beginning postnatal day (PND) 28 is associated with accelerated puberty onset in female rats. We also found a reduction in the number and activity of (hypocretin)-producing neurons in hypothalamus of BPA rats, which was associated with broad motivational deficits. Because BPA induces neuroinflammation throughout the brain, here we tested if changes in orexin levels might be associated with changes in local microglia number and morphology. Female rats (n=8/group) were exposed to BPA (0, 25, 250ug/kg/d) via their drinking water from PND28-56, sacrificed, and their brains sectioned for immunohistochemical detection of orexin-containing neurons and microglia. As in our previous studies, BPA treated rats had a decreased number of orexin-containing neurons compared to controls (p<0.05). This was associated with an increase in the number of activated-state microglia (p<0.01), and increased contacts between microglia and orexin neurons (p<0.05, ANOVA and Tukey’s post-hoc test). These data indicate that the alterations in behavior and orexin expression in our previous studies may be mediated by neuroinflammation. Thus, strategies that reduce neuroinflammation might protect against the consequences of BPA exposure on orexin system function and associated behaviors.

 

Addition Research (AR)

AR1. MIR-129 AND MIR-182 SUPPRESS AGGRESSIVENESS OF FETAL ALCOHOL INDUCED PITUITARY NEUROENDOCRINE TUMORS
D. Sarkar, O. Gangisetty, S. Chaudhary
Endocrine Research Program, Department of Animal Science, Rutgers University, New Brunswick, NJ-08901

Pituitary neuroendocrine tumors (PitNETs) are characterized by high vascularization, grass invasion of surrounding tissue and metastasis. These tumors often lack sensitivity to therapy or recur after treatment. We have recently shown that fetal alcohol exposure (FAE) promotes the development of PitNETs. The mechanism by which fetal alcohol promotes PitNETs is not clearly understood. MicroRNAs are known to participate in the development of aggressive PitNETs. Whether microRNAs participate in FAE induced increase in development of aggressive PitNETs is studied. We used pituitary tumor cells derived from rats fed with alcohol (AF) or control rat chow (AD) during fetal period (postnatal day 7-21) and treated with estradiol for 3 months during adult. AF pituitary cells showed different cell morphology, increased cell proliferation, cell migration and colony formation as compared to AD cells, suggesting that AF cells have increased aggressiveness features than those of AD cells. Taqman based TILDA array was employed to determine the microRNA expression profiles and identified altered expressions of a number of microRNAs in AF cells compared to AD cells. Genome wide miRNA expression profile also identified three different microRNAs; miR-129, miR-182 and miR-499 were down regulated in AF cells compared to AD cells. The reduced expression of these three miRNAs was further confirmed by Taqman miRNA assay data. Overexpression of miRNA, using mimic oligo transfection in AF cells, we found that miR-129,182 or 499 mimic oligo robustly increased the expression of corresponding miRNAs. We also measured cell proliferation, cell migration and colony formation in AF cells transfected with miRNA mimic oligos. Our results show that miR-129 or miR182 mimic oligos significantly reduced cell proliferation, cell migration and number of colonies formed compared to the negative control oligos suggesting that these two miRNAs play a role in suppressing the tumor characteristic feature of AF cells. However, miR-499 mimic oligo had no effect on cell proliferation, colony formation and cell migration. Bioinformatic tools predicted target genes for these two miRNAs include Sox family members, Wnts and MMPs. We further evaluated the role of these two miRNAs in regulation of the target genes in AF rat pituitary cells. Our results suggest that miR-129 or 182 may serve as tumor suppressor miRNAs in AF rat pituitary tumor cells. (Supported by NIH grant R01 AA 11591).

AR2. Treatment with ethanol upregulation of MTOR and TP53 in U87MG cells
S. Chang, A. Hassan, J. Zhang, W. Huang
Seton Hall University Department of Biological Sciences, Seton Hall University Institute of NeuroImmune Pharmacology and Department of Biological Sciences

Alcohol is known as a carcinogen. Alcohol misuse and alcohol use disorders augment brain pathologies, including those associated with glioblastoma multiform (GBM) that is a deadly brain tumor of adults. Some studies have been done regarding involvement of alcohol use in GBM development. However, those studies have been tangled in uncertainties and no attempt was made to reveal mechanisms underlying ethanol (EtOH) enhancement of GBM progression. By integrating data-driven discovery and hypothesis testing investigation we hope to address this challenge. The network algorithm of Ingenuity Pathway Analysis was employed in using QIAGEN Knowledge Base. Three meta-analyses have further confirmed our hypothesis “binge EtOH augmentation of GBM progression”: 1) EtOH activated microglial migration, 2) EtOH enhanced development of GBM, 3) EtOH enhanced GBM pathology via STAT3 as an upstream regulator and intermediary molecules miR-21, CCNE1, BNP3L, VEGFA, WNTSA, SRC, cyclin D, miR-29, MTOR, CXCR4, TP53, PRKCD, PROM1. Based on these discoveries, we have identified five focus genes in EtOH enhanced GBM progression, PROM1, PRKCD, TP53, CXCR4 and MTOR. U87MG was used to study EtOH modulation of expression of these 5 genes. The cells were treated with EtOH at 0, 17.4 or 52.2 mM for either 8h or 24h. Following treatment, the cells were harvested. The cell number and viability were determined. RNA was isolated for cDNA preparation for qRT-PCR. Treatment with EtOH for 24 hr decreased U87MG cell number and viability. The data of qRT-PCR showed a trend of increased expression of MTOR and TP53 following treatment with EtOH for 24 hr while alternation of PRKCD expression to treatment with EtOH was not observed. PROM1 expression was not detected and expression of CXCR4 in U87MG cells was very low. Taken together, our in-vitro studies have revealed that treatment with EtOH might enhance GBM progression by upregulating expression of MTOR and TP53.

AR3. Chronic Pain in Comorbid Alcohol Use Disorder and HIV
S. Chang, M.S.T.A. Azeeze, M. Bishir
Institute of NeuroImmune Pharmacology, Seton Hall University, South Orange, NJ, 07079, USA and Department of Biological Sciences, Seton Hall University, South Orange, NJ, 07079, USA.

Alcohol misuse is highly prevalent among HIV patients. The virus and alcohol interact through multiple mechanisms, amplifying the negative effects, including exacerbation of pain. We have previously shown that short-term alcohol exposure produced analgesic effect, while prolonged exposure results in chronic pain. HIV-patients often experience pain and sometimes use alcohol to self-medicate alcohol to combat pain. A little is known about the combined impact of alcohol and HIV on pain. We hypothesize that alcohol exposure enhances pain responses to HIV virus. In this study, we analyzed differentially expressed genes (DEGs) from three GSE datasets to study how alcohol exposure enhances the pain in HIV subjects: 1) GSE69685 Macaca mulatta, exposed to binge ethanol (EtOH) and SIV infection; 2) GSE47474 HIV-1Tg rats, possessing HIV viral proteins without viral replication; and 3) GSE28160 post-mortem brain samples from HIV patients. Ingenuity Pathway Analysis (IPA)-Core analysis of the DEGs from Macaca mulatta hippocampus revealed the activation of neuroinflammation, neuropathic pain signaling pathways, and the inhibition of opioid signaling. The DEGs were also involved in the increase of neuromuscular disease with neuropathy. DEGs from GSE47474 (HIV viral proteins) and GSE28160 (HIV virus) showed similar enrichment of these three signaling pathways, with DEGs from GSE28160 alone showed an increase in neuromuscular disease with neuropathy. To examine the impact of alcohol exposure onto these DEGs, we utilized IPA tools to simulate EtOH, mimicking alcohol exposure. The EtOH simulation resulted in the enhancement of discomfort in DEGs from HIV-1Tg rats and increased neuromuscular disease with neuropathy in DEGs from HIV patients. We also found a significant overlap between the DEGs from the Macaca mulatta, Human and HIV-1 Tg rat GSE datasets. EtOH simulation of these DEGs also showed an increase in discomfort. Taken together, utilizing data-driven approaches, we demonstrated pain in comorbid alcohol misuse and HIV infection. Keywords: Neuroinflammation, Neuropathy, Differentially Expressed Genes, Gene Expression Omnibus, Qiagen Knowledge Base, Ingenuity Pathway Analysis.