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Maternal genotype and high fat feeding in utero program hyperglycemia and risk for cardiovascular disease
S10 SMFM Abstracts 22
INCREASED SMALL GTPASES- THE FUNDAMENTAL MOLECULAR MECHANISM UNDERLYING DECREASED MYOMETRIAL RESPONSIVENESS DURING QUIESCENCE CLIFFORD MASON1, PETER W. SWAAN1, CARL P. WEINER2, 1University of Maryland at Baltimore, Pharmaceutical Sciences, Baltimore, Maryland, 2University of Maryland at Baltimore, Physiology and Obstetrics, Gynecology and Reproductive Sciences, Baltimore, Maryland OBJECTIVE: The proposed mechanisms of myometrial quiescence include progesterone, fewer gap junctions, altered receptor density, etc. We showed that guinea pig (GP) myometrial quiescence was associated with increased GTPase activity. Given their pivotal role modulating secondary effector responses after receptor activation, we speculated the increased GTPase activity could provide a unifying pathway for decreased receptor mediated responses by reducing the half-life of the activated receptor complex. The object of this study was to identify the mechanism for increased myometrial GTPase activity. STUDY DESIGN: Gene profiling was performed with myometrial RNA obtained from nonpregnant, midpregnant (MP, w0.67 gestation), termpregnant (TP, w0.96 gestation), castrate and estradiol-treated castrate GPs (Affymetrix Human Genome U133A). Gene expression was normalized using Affymetrix Microarray Suite 5.0. Fold-changes in levels were calculated from the mean signals during MP and TP vs. their NP controls. Cluster analysis was performed by separating the genes into those up- and down-regulated during each time period using MetaCoreÔ. Each category was further subdivided into genes specific for quiescence and activation. RESULTS: The impact of pregnancy on G-protein related mRNA was modest. Only the small GTPase Rap1b showed altered expression O2 fold (up 3! during quiescence) across gestation. But network analyses revealed that only Rab7, another small GTPase was exclusively upregulated (80%) during quiescence. In contrast, network analysis showed G-protein as the only component exclusively upregulated during late pregnancy. Estradiol mimicked the effects of pregnancy on some but not all potentially relevant G-protein family genes (including Rap1). CONCLUSION: The increased MP myometrial GTPase activity appears at least in part secondary to increased transcription of the small GTPases Rap1b and Rab7. The application of computer annotated databases allowed for the visualization of ‘pregnancy’ and ‘estradiol’ networks and universes that provide a basis for future investigation.
24
MATERNAL GENOTYPE AND HIGH FAT FEEDING IN UTERO PROGRAM HYPERGLYCEMIA AND RISK FOR CARDIOVASCULAR DISEASE ESTHER SCHMUEL (F)1, KIRSTEN HARTIL2, PATRICIA VUGUIN2, MATTHEW WARNER2, CARLOS VARGAS2, MAUREEN CHARRON2, 1 Albert Einstein College of Medicine, Obstetrics & Gynecology and Women’s Health, Bronx, New York, 2Albert Einstein College of Medicine, Deparment of Biochemisty, Bronx, New York OBJECTIVE: To determine the effects of in utero high fat diet (HFD) and maternal genotype on the metabolic programming of offspring. STUDY DESIGN: Female CD1 mice aged 10-12 weeks, either wild type (WT) or glucose transporter-4 heterozygous knockout (G4) (genetic model of insulin resistance), were fed a HFD (36% fat) or control breeding chow (10% fat) for 2 weeks prior to mating, through pregancy and lactation. WT male offspring were weaned onto low-fat diet (5% fat). Four groups were analyzed: offspring of WT mothers on HFD [WT-HFD] or control chow [WT-C], and offspring of G4 mothers on HFD [G4-HFD] or control chow [G4-C], (n=8-14/group). Fed and fasted glucose, insulin, and adipokines were analyzed. At sacrifice (24 weeks) organ weights were recorded. Statistical analysis was performed using ANOVA and two tailed t test. RESULTS: Maternal glucose levels, litter size, and birth weights were similar in all groups. At sacrifice, body weight (BW) was higher in WT-HFD compared to G4-HFD (48 vs. 41 gm, p=.03). Fed and fasted glucose levels were higher in WT-HFD compared to WT-C (224.5 vs 146.5 mg/dL, p=.02; 128.5 vs 106.8, p=.04). Fed insulin levels were similar in all groups. Serum adiponectin was 30-40% lower in WT-HFD (4 vs 5.8 pg/mL, p=.002) and G4HFD (4 vs 6.5 pg/mL, p=.001) compared to respective controls. PAI-1 and IL-6, markers of systemic inflammation, were elevated in WT-HFD compared to WT-C (2538.83 vs. 820.00 pg/mL, p=.001) but not in G4-HFD and G4-C. At sacrifice, liver/BW and kidney/BW ratios were higher in WT-HFD compared to WT-C (p=.01 and p=.009) but not different in G4-HFD compared to G4-C. CONCLUSION: Offspring of WT-HFD females exhibit hyperglycemia, alterations in adipokine profiles, and evidence of systemic inflammation that may increase cardiovascular risk. In contrast, offspring of G4 females appear protected from this programming effect.
23
BLOOD PRESSURE REGULATION AND UTERINE ENVIRONMENT IN CONSCIOUS UNRESTRAINED NOS3 KNOCKOUT MICE MONICA LONGO1, EGLE BYTAUTIENE1, FANGXIAN LU1, ESTHER TAMAYO1, GARY HANKINS1, GARLAND ANDERSON1, GEORGE SAADE1, 1University of Texas Medical Branch at Galveston, Obstetrics and Gynecology, Galveston, Texas OBJECTIVE: Nitric oxide, produced mainly by endothelial nitric oxide synthase (NOS3), has been implicated in the vascular adaptations to pregnancy, and its deficiency has been linked to uteroplacental insufficiency and preeclampsia in some but not all studies. All animal studies, however, have been performed in anesthetized or restrained animals, or in animals treated with various inhibitors, thereby introducing confounding variables. Our aim was to determine the role of NOS3 in pregnancy using unrestrained transgenic mice. STUDY DESIGN: At day 10 of gestation, blood pressure (BP) catheters connected to telemetric transducers were inserted through the left carotid artery into the aortic arch of homozygous NOS3 knockout (C57BL/6JNOS3ÿ/ÿKO) and wild type mice (NOS3C/CWT). BP was recorded telemetrically and continuously in the conscious unrestrained animals from day 12 until day 18 of gestation. At day 18, the mice were sacrificed, and the pups and placentas were weighed. BP mean was calculated for each day of gestation. Student’s t-test and repeated measures 2-way ANOVA were used for statistical analysis (significance: p!0.05). RESULTS: Mean BP in NOS3ÿ/ÿKO was significantly higher from day 12 of gestation until day 18 (D12: 145.4G7.4 and D18: 127.4G1.1 mmHg) compared with NOS3C/CWT (D12: 98.1G2.4 and D18: 95.1G3.1 mmHg). The average pup weight (0.82G0.04 vs 2.28G0.43 g) and placental weight (0.11G0.01 vs 0.15G0.01 g) were significantly lower in the NOS3ÿ/ÿKO compared with NOS3C/CWT. CONCLUSION: This is the first evidence that conscious, unrestrained, transgenic mice lacking NOS3 are hypertensive throughout later gestation, and have evidence of uteroplacental insufficiency. Our findings support a role for nitric oxide and NOS3 in the vascular adaptations to pregnancy, and a role for its deficiency in hypertensive disorders of pregnancy and abnormal uterine environment.
25
PROGESTERONE REDUCES LIPOPOLYSACCHARIDE INDUCED INFLAMMATORY CYTOKINE SECRETION IN FETOPLACENTAL ARTERIES AND FRACTIONATED CORD BLOOD JENNIFER "GOTKIN (F)1, PATRICK MCNUTT2, JEREMY CELVER2, ANDREA SHIELDS1, JIM WRIGHT2, PETER NAPOLITANO1, 1Madigan Army Medical Center, Obstetrics and Gynecology, Tacoma, Washington, 2Madigan Army Medical Center, Department of Clinical Investigation, Tacoma, Washington OBJECTIVE: Progesterone (P4) therapy is currently used for prevention of recurrent preterm delivery, however, its physiologic mechanism has yet to be described. Our prior work demonstrates P4 treatment of fetoplacental vascular tissue decreases lipopolysaccharide (LPS)-induced production of Interleukin-6 (IL-6). This study characterizes the effects of P4 on IL-6 production by fractionated leukocytes, granulocytes, whole blood and fetoplacental artery endothelium. STUDY DESIGN: Tissues were collected from 3 placentas obtained from healthy term pregnancies undergoing non-labored repeat cesarean section. Chorionic plate arteries were washed then dissected, and fetal cord blood was either diluted 1:10 in culture media or fractionated by histopaque gradient into granulocyte and mononuclear populations. All fractions were then incubated in Dulbecco’s modified Eagle’s medium supplemented with either LPS (50 ug/ mL), P4 (30ng/mL) or pretreated with P4 for 24 hours and then exposed to LPS for 16 hours. Culture supernatants were collected and evaluated for secreted IL-6 by ELISA. Data was analyzed using Student’s t test, P value ! 0.05 significant. RESULTS: Exposure of endothelium and mononuclear cells to LPS increased IL-6 secretion by 23-fold (p!0.005) and 37-fold (p!0.001), respectively, while granulocytes were non-responsive. Pretreatment with P4 decreased endothelial IL-6 secretion by 7-fold (p!0.001) and mononuclear cell secretion by 2-fold (p!0.005). Although whole blood exhibited LPSinduced IL-6 production, the net changes were consistent with an additive effect proportional to the above subpopulations. CONCLUSION: Inflammatory cytokine production strongly correlates with preterm labor. We found tissue-specific response to P4 is consistent with expected distribution of progesterone receptors. Our study suggests one mechanism by which P4 may prevent preterm delivery is tissue-specific inhibition of IL-6 production. This model will be used to develop proteomic-based methods to elucidate molecular events underlying P4-mediated inhibition of inflammatory cytokine production.
INCREASED SMALL GTPASES- THE FUNDAMENTAL MOLECULAR MECHANISM UNDERLYING DECREASED MYOMETRIAL RESPONSIVENESS DURING QUIESCENCE CLIFFORD MASON1, PETER W. SWAAN1, CARL P. WEINER2, 1University of Maryland at Baltimore, Pharmaceutical Sciences, Baltimore, Maryland, 2University of Maryland at Baltimore, Physiology and Obstetrics, Gynecology and Reproductive Sciences, Baltimore, Maryland OBJECTIVE: The proposed mechanisms of myometrial quiescence include progesterone, fewer gap junctions, altered receptor density, etc. We showed that guinea pig (GP) myometrial quiescence was associated with increased GTPase activity. Given their pivotal role modulating secondary effector responses after receptor activation, we speculated the increased GTPase activity could provide a unifying pathway for decreased receptor mediated responses by reducing the half-life of the activated receptor complex. The object of this study was to identify the mechanism for increased myometrial GTPase activity. STUDY DESIGN: Gene profiling was performed with myometrial RNA obtained from nonpregnant, midpregnant (MP, w0.67 gestation), termpregnant (TP, w0.96 gestation), castrate and estradiol-treated castrate GPs (Affymetrix Human Genome U133A). Gene expression was normalized using Affymetrix Microarray Suite 5.0. Fold-changes in levels were calculated from the mean signals during MP and TP vs. their NP controls. Cluster analysis was performed by separating the genes into those up- and down-regulated during each time period using MetaCoreÔ. Each category was further subdivided into genes specific for quiescence and activation. RESULTS: The impact of pregnancy on G-protein related mRNA was modest. Only the small GTPase Rap1b showed altered expression O2 fold (up 3! during quiescence) across gestation. But network analyses revealed that only Rab7, another small GTPase was exclusively upregulated (80%) during quiescence. In contrast, network analysis showed G-protein as the only component exclusively upregulated during late pregnancy. Estradiol mimicked the effects of pregnancy on some but not all potentially relevant G-protein family genes (including Rap1). CONCLUSION: The increased MP myometrial GTPase activity appears at least in part secondary to increased transcription of the small GTPases Rap1b and Rab7. The application of computer annotated databases allowed for the visualization of ‘pregnancy’ and ‘estradiol’ networks and universes that provide a basis for future investigation.
24
MATERNAL GENOTYPE AND HIGH FAT FEEDING IN UTERO PROGRAM HYPERGLYCEMIA AND RISK FOR CARDIOVASCULAR DISEASE ESTHER SCHMUEL (F)1, KIRSTEN HARTIL2, PATRICIA VUGUIN2, MATTHEW WARNER2, CARLOS VARGAS2, MAUREEN CHARRON2, 1 Albert Einstein College of Medicine, Obstetrics & Gynecology and Women’s Health, Bronx, New York, 2Albert Einstein College of Medicine, Deparment of Biochemisty, Bronx, New York OBJECTIVE: To determine the effects of in utero high fat diet (HFD) and maternal genotype on the metabolic programming of offspring. STUDY DESIGN: Female CD1 mice aged 10-12 weeks, either wild type (WT) or glucose transporter-4 heterozygous knockout (G4) (genetic model of insulin resistance), were fed a HFD (36% fat) or control breeding chow (10% fat) for 2 weeks prior to mating, through pregancy and lactation. WT male offspring were weaned onto low-fat diet (5% fat). Four groups were analyzed: offspring of WT mothers on HFD [WT-HFD] or control chow [WT-C], and offspring of G4 mothers on HFD [G4-HFD] or control chow [G4-C], (n=8-14/group). Fed and fasted glucose, insulin, and adipokines were analyzed. At sacrifice (24 weeks) organ weights were recorded. Statistical analysis was performed using ANOVA and two tailed t test. RESULTS: Maternal glucose levels, litter size, and birth weights were similar in all groups. At sacrifice, body weight (BW) was higher in WT-HFD compared to G4-HFD (48 vs. 41 gm, p=.03). Fed and fasted glucose levels were higher in WT-HFD compared to WT-C (224.5 vs 146.5 mg/dL, p=.02; 128.5 vs 106.8, p=.04). Fed insulin levels were similar in all groups. Serum adiponectin was 30-40% lower in WT-HFD (4 vs 5.8 pg/mL, p=.002) and G4HFD (4 vs 6.5 pg/mL, p=.001) compared to respective controls. PAI-1 and IL-6, markers of systemic inflammation, were elevated in WT-HFD compared to WT-C (2538.83 vs. 820.00 pg/mL, p=.001) but not in G4-HFD and G4-C. At sacrifice, liver/BW and kidney/BW ratios were higher in WT-HFD compared to WT-C (p=.01 and p=.009) but not different in G4-HFD compared to G4-C. CONCLUSION: Offspring of WT-HFD females exhibit hyperglycemia, alterations in adipokine profiles, and evidence of systemic inflammation that may increase cardiovascular risk. In contrast, offspring of G4 females appear protected from this programming effect.
23
BLOOD PRESSURE REGULATION AND UTERINE ENVIRONMENT IN CONSCIOUS UNRESTRAINED NOS3 KNOCKOUT MICE MONICA LONGO1, EGLE BYTAUTIENE1, FANGXIAN LU1, ESTHER TAMAYO1, GARY HANKINS1, GARLAND ANDERSON1, GEORGE SAADE1, 1University of Texas Medical Branch at Galveston, Obstetrics and Gynecology, Galveston, Texas OBJECTIVE: Nitric oxide, produced mainly by endothelial nitric oxide synthase (NOS3), has been implicated in the vascular adaptations to pregnancy, and its deficiency has been linked to uteroplacental insufficiency and preeclampsia in some but not all studies. All animal studies, however, have been performed in anesthetized or restrained animals, or in animals treated with various inhibitors, thereby introducing confounding variables. Our aim was to determine the role of NOS3 in pregnancy using unrestrained transgenic mice. STUDY DESIGN: At day 10 of gestation, blood pressure (BP) catheters connected to telemetric transducers were inserted through the left carotid artery into the aortic arch of homozygous NOS3 knockout (C57BL/6JNOS3ÿ/ÿKO) and wild type mice (NOS3C/CWT). BP was recorded telemetrically and continuously in the conscious unrestrained animals from day 12 until day 18 of gestation. At day 18, the mice were sacrificed, and the pups and placentas were weighed. BP mean was calculated for each day of gestation. Student’s t-test and repeated measures 2-way ANOVA were used for statistical analysis (significance: p!0.05). RESULTS: Mean BP in NOS3ÿ/ÿKO was significantly higher from day 12 of gestation until day 18 (D12: 145.4G7.4 and D18: 127.4G1.1 mmHg) compared with NOS3C/CWT (D12: 98.1G2.4 and D18: 95.1G3.1 mmHg). The average pup weight (0.82G0.04 vs 2.28G0.43 g) and placental weight (0.11G0.01 vs 0.15G0.01 g) were significantly lower in the NOS3ÿ/ÿKO compared with NOS3C/CWT. CONCLUSION: This is the first evidence that conscious, unrestrained, transgenic mice lacking NOS3 are hypertensive throughout later gestation, and have evidence of uteroplacental insufficiency. Our findings support a role for nitric oxide and NOS3 in the vascular adaptations to pregnancy, and a role for its deficiency in hypertensive disorders of pregnancy and abnormal uterine environment.
25
PROGESTERONE REDUCES LIPOPOLYSACCHARIDE INDUCED INFLAMMATORY CYTOKINE SECRETION IN FETOPLACENTAL ARTERIES AND FRACTIONATED CORD BLOOD JENNIFER "GOTKIN (F)1, PATRICK MCNUTT2, JEREMY CELVER2, ANDREA SHIELDS1, JIM WRIGHT2, PETER NAPOLITANO1, 1Madigan Army Medical Center, Obstetrics and Gynecology, Tacoma, Washington, 2Madigan Army Medical Center, Department of Clinical Investigation, Tacoma, Washington OBJECTIVE: Progesterone (P4) therapy is currently used for prevention of recurrent preterm delivery, however, its physiologic mechanism has yet to be described. Our prior work demonstrates P4 treatment of fetoplacental vascular tissue decreases lipopolysaccharide (LPS)-induced production of Interleukin-6 (IL-6). This study characterizes the effects of P4 on IL-6 production by fractionated leukocytes, granulocytes, whole blood and fetoplacental artery endothelium. STUDY DESIGN: Tissues were collected from 3 placentas obtained from healthy term pregnancies undergoing non-labored repeat cesarean section. Chorionic plate arteries were washed then dissected, and fetal cord blood was either diluted 1:10 in culture media or fractionated by histopaque gradient into granulocyte and mononuclear populations. All fractions were then incubated in Dulbecco’s modified Eagle’s medium supplemented with either LPS (50 ug/ mL), P4 (30ng/mL) or pretreated with P4 for 24 hours and then exposed to LPS for 16 hours. Culture supernatants were collected and evaluated for secreted IL-6 by ELISA. Data was analyzed using Student’s t test, P value ! 0.05 significant. RESULTS: Exposure of endothelium and mononuclear cells to LPS increased IL-6 secretion by 23-fold (p!0.005) and 37-fold (p!0.001), respectively, while granulocytes were non-responsive. Pretreatment with P4 decreased endothelial IL-6 secretion by 7-fold (p!0.001) and mononuclear cell secretion by 2-fold (p!0.005). Although whole blood exhibited LPSinduced IL-6 production, the net changes were consistent with an additive effect proportional to the above subpopulations. CONCLUSION: Inflammatory cytokine production strongly correlates with preterm labor. We found tissue-specific response to P4 is consistent with expected distribution of progesterone receptors. Our study suggests one mechanism by which P4 may prevent preterm delivery is tissue-specific inhibition of IL-6 production. This model will be used to develop proteomic-based methods to elucidate molecular events underlying P4-mediated inhibition of inflammatory cytokine production.