Research supported by NIH HD070096-01A1 Maternal obesity induces increased placental conversion of maternal cortisol to cortisone followed by increased conversion of this cortisone to cortisol by the fetal liver and perirenal fat Ashley M. Smith, Adel B. Ghnenis, J. Fred Odhiambo, Peter W. Nathanielsz and Stephen P. Ford. Center for the Study of Fetal Programming, Department of Animal Science, University of Wyoming, Laramie, WY, USA; Email:Asmit121@uwyo.edu Abstract Elevated maternal and fetal blood cortisol concentrations are seen from mid to late gestation with maternal obesity in the ewe, and have been associated with adverse postnatal health effects on offspring including hyperphagia, increased weight gain, adiposity, and increased insulin resistance. Further, this maternal obesity associated fetal cortisol increase occurs in the absence of a corresponding increase in fetal ACTH release suggesting the possibility of a nonadrenal source. This study was conducted to determine the source of this fetal cortisol rise, and whether it was of fetal or maternal origin. Multiparous ewes were fed either a control (CTR, n=7) diet at 100% National Research Council (NRC) recommendations, or an obesogenic (OB, n=7) diet at 150% NRC. Experimental diets started 60 days before conception and continued through necropsy on day 135 of gestation. Under isoflurane anesthesia, maternal jugular venous and umbilical venous blood were collected, and following exsanguination of the ewe and fetus, placental (cotyledonary) and fetal (liver and perirenal fat) tissues were harvested and snap frozen in liquid nitrogen. A validated radioimmunoassay was conducted to determine cortisol concentrations in maternal and fetal blood. Western blots were conducted on fetal liver and perirenal fat as well as cotyledonary tissue to quantify protein expression of three key genes involved in cortisol metabolism: 1) 11β hydroxysteroid dehydrogenase 1 (11β-HSD1) which catalyzes the conversion of cortisone to cortisol, 2) hexose-6-phosphate dehydrogenase (H6PD) which generates the NADPH required for 11β-HSD1 to convert cortisone into cortisol, and 3)11β-HSD2 which converts cortisol to cortisone. Group differences were determined via the proc MIXED procedure of SAS. Cortisol concentrations were greater in jugular venous blood of OB compared to CTR ewes (79.10±8.57 vs. 19.86±6.78 ng/ml, P<0.001) and in umbilical venous blood of OB compared to CTR fetuses (45.51±5.07 vs. 23.69±5.48 ng/ml, P<0.01) on day 135 of gestation. The expression of 11β-HSD2 was higher (P<0.05) in OB cotyledonary tissue than CTR cotyledonary tissue (0.51±0.045 vs. 0.37±0.04 arbitrary units [au]), while no group differences were found in cotyledonary tissue expression of 11β-HSD1. In contrast, 11β-HSD1 expression was increased (P<0.05) in both OB versus CTR fetal liver (0.70±0.13 vs.0.27±0.13 au,), and perirenal fat (1.72±0.17 vs 1.19±0.17 au). H6PD tended to increase (P<0.10) in the liver of OB versus CTR fetuses (0.65±0.06 vs. 0.48±0.05 au) and was increased (P<0.05) in OB vs. CTR perirenal fat (0.40±0.03 vs 0.29±0.03 au). No group differences in 11β-HSD2 were found in either liver or perirenal fat. These data suggest that the increased OB cotyledonary 11β-HSD2 may convert increased maternal cortisol into cortisone before entering the fetal compartment. Further, the increased circulating cortisone in OB fetuses may then be used to synthesize increased cortisol concentrations by the fetal liver and perirenal fat. These data provide evidence that maternal obesity elevates cortisol production through conversion of cortisone to cortisol within the fetal compartment, which could predispose offspring to metabolic dysfunction in postnatal life. Figure 1: Glucocorticoid metabolite concentrations on day 135 of gestation of (A.) cortisol in maternal plasma (B.) cortisone in fetal plasma (C.) cortisol in fetal plasma. Means ± SEM Differ; *P<0.01; **P<0.001. A. B. C. n = 7 per group n = 12 per group Materials and Methods Nonpregnant multiparous, white-faced ewes were randomly assigned to one of two dietary groups, Control (CTR) or Obese (OB). The CTR group received 100% of National Research Council (NRC) recommendations, while the OB group received 150% of NRC recommendations from 60 days prior to conception to necropsy, performed on day 135 of gestation (0.9 of total gestation). Immediately prior to exsanguination, maternal jugular venous blood and fetal umbilical venous blood were collected into 10mL heparinized tubes and plasma stored at -80°C. Selected fetal organs were harvested, snap frozen in liquid nitrogen and then stored at -80°C. Fully validated radioimmunoassays were utilized to determine cortisol concentrations in maternal plasma as well as cortisol and cortisone concentrations in fetal plasma. Protein expression of 11β-HSD2, 11β-HSD1 and H6PD within cotyledonary tissue, fetal perirenal fat and fetal liver were determined via Western Blot Analysis. Results were statistically analyzed using the PROC MIXED procedure in SAS. Figure 2: Protein expression of (A.) 11β-HSD1 and (B.) 11β-HSD2 within placental (cotyledonary) tissue on day 135 of gestation. Means ± SEM Differ; *P<0.05. 11β-HSD 1 β-Actin n = 7 per group 11β-HSD 2 A. B. Results Maternal and fetal cortisol and fetal cortisone concentrations were significantly elevated in OB vs. CTR animals on day 135 of gestation; with a positive correlation between OB ewe plasma cortisol and OB fetal plasma cortisone levels (r=0.8, P-value<0.01, r2 =0.64). There was a significant upregulation of 11β-HSD2 within OB vs. CTR placental (cotyledonary) tissue on day 135 of gestation, consistent with an increased conversion of maternal cortisol into fetal cortisone. There was a significant upregulation of 11β-HSD1, and its co-factor H6PD, in both fetal perirenal adipose tissue and liver of OB vs. CTR fetuses on day 135 of gestation; with a positive correlation between OB fetal plasma cortisone and OB fetal plasma cortisol levels (r=0.7, P-value<0.0001, r2 =0.51). Figure 3: Protein expression of (A.) 11β-HSD1, (B.) the co-factor H6PD and (C.) 11β-HSD2 within fetal liver on day 135 of gestation. Means ± SEM Differ; *P<0.05, #P<0.07. 11β-HSD 1 β-Actin H6PD 11β-HSD 2 A. B. C. n = 7 per group Conclusion These results are consistent with the concept that maternal obesity results in elevated extra-adrenal fetal cortisol production, leading to higher concentrations of this hormone in fetal blood. These data suggest there is an upregulation of 11β-HSD2 within the placenta (cotyledonary tissue) of OB ewes to convert elevated maternal cortisol into fetal cortisone. This increased circulating cortisone, along with an upregulation of 11β-HSD1 and the co-factor H6PD, is then used by OB fetal perirenal adipose tissue and liver to synthesize and release increased concentrations of cortisol into fetal circulation, possibly predisposing offspring to metabolic dysfunction in postnatal life. This cascade of events is depicted below. Introduction The release of cortisol, the main glucocorticoid produced by the zona fasciculata of the adrenal cortex, is normally controlled via the hypothalamic-pituitary-adrenal axis in response to stress and low blood glucose concentrations. Chronic elevation of blood cortisol, however, can influence a cadre of adverse health effects including, obesity, hypertension and insulin resistance, which are known components of the metabolic syndrome. Our lab has previously shown that maternally obese (OB) ewes along with their developing fetuses have elevated blood levels of cortisol from mid (day 75) to late (day 135) gestation when compared to control fed (CTR) ewes and fetuses. While the chronic elevation of maternal cortisol of OB ewes was associated with an elevation in blood adrenocorticotropic hormone (ACTH), the rise in fetal blood cortisol was not, suggesting an extra-adrenal source of cortisol production. Placental 11β hydroxysteroid dehydrogenase (11β-HSD type 2) is the enzyme that converts active maternal cortisol to inactive fetal cortisone. In contrast, 11β-HSD1, which is highly expressed in the fetal liver and perirenal adipose tissue, facilitates the conversion of inactive cortisone into active cortisol, in the presence of the co-factor, hexose -6- phosphate dehydrogenase (H6PD). It is possible that an OB-induced increase in both placental 11β-HSD2 (would increase circulating fetal cortisone) and increases in liver and perirenal adipose tissue levels of 11β-HSD1 plus H6PD (increases conversion of this elevated cortisone to cortisol) might mediate the chronic increase in circulating cortisol seen in fetuses of obese ewes. Alterations of 11β-HSD1 have been suggested to play a role in the pathogenesis of the metabolic syndrome. H6PD n = 7 per group 11β-HSD 2 β-Actin C. A. B. Study Objective The objective of this study was to measure protein expression changes in placental 11β-HSD2 and liver and perirenal adipose tissue levels of 11β-HSD1 plus H6PD in fetuses gestated by OB and CTR ewes. n = 7 per group 11β-HSD 1 β-Actin n = 7 per group H6PD β-Actin Figure 4: Protein expression of (A.) 11β-HSD1, (B.) the co-factor H6PD and (C.) 11β-HSD2 within fetal perirenal adipose tissue on day 135 of gestation. Means ± SEM differ; *P<0.05. Research supported by NIH HD070096-01A1