Kanchan M. Kaushal

Long-term hypoxia modulates expression of key genes regulating adipose function in the late-gestation ovine fetus

A major function of abdominal adipose in the newborn is nonshivering thermogenesis. Uncoupling protein (UCP) UCP1 and UCP2 play major roles in thermogenesis. The present study tested the hypothesis that long-term hypoxia (LTH) modulates expression of UCP1 and UCP2, and key genes regulating expression of these genes in the late-gestation ovine fetus. Ewes were maintained at high altitude (3,820 m) from 30 to 138 days gestation (dG); perirenal adipose tissue was collected from LTH and age-matched, normoxic control fetuses at 139-141 dG. Quantitative real-time PCR was used to analyze mRNA for UCP1, UCP2, 11beta hydroxysteroid dehydrogenase type 1 (HSD11B1) and 2 (HSD11B2), glucocorticoid receptor (GR), beta3 adrenergic receptor (beta3AR), deiodinase type 1 (DIO1) and DIO2, peroxisome proliferator activated receptor (PPAR) alpha and gamma and PPARgamma coactivator 1 (PGC1alpha). Concentrations of mRNA for UCP1, HSD11B1, PPARgamma, PGC1, DIO1, and DIO2 were significantly higher in perirenal adipose of LTH compared with control fetuses, while mRNA for HSD11B2, GR, or PPARalpha in perirenal adipose did not differ between control and LTH fetuses. The increased expression of UCP1 is likely an adaptive response to LTH, assuring adequate thermogenesis in the event of birth under oxygen-limiting conditions. Because both glucocorticoids and thyroid hormone regulate UCP1 expression, the increase in HSD11B1, DIO1, and DIO2 implicate increased adipose capacity for local synthesis of these hormones. PPARgamma and its coactivator may provide an underlying mechanism via which LTH alters development of the fetal adipocyte. These findings have important implications regarding fetal/neonatal adipose tissue function in response to LTH.

Circadian myometrial and endocrine rhythms in the pregnant rhesus macaque: Effects of constant light and timed melatonin infusion

Six chronically catheterized rhesus macaques maintained on a 12-hour-light/dark cycle (lights on from 7 AM to 7 PM) showed a nocturnal uterine activity rhythm with peak contractile events between 9 and 11 PM (p less than 0.05). In blood samples collected at 3-hour intervals over a 24-hour period, we determined that plasma melatonin and progesterone concentrations were elevated at night whereas estradiol, estrone, and cortisol reached peak concentrations in the early morning (p less than 0.05). Lights were then left on for the remainder of the study. After 12 days in constant light, daily rhythms in uterine activity and plasma steroid levels were relatively unchanged, whereas melatonin concentrations were suppressed. Animals then received a timed infusion of melatonin (0.2 mg/kg/hr each day from 7 PM to 6 AM daily until delivery). The nocturnal uterine activity rhythm and the rhythms in plasma steroid concentrations were maintained. We conclude that the 24-hour patterns in maternal uterine activity and plasma steroid hormone levels are circadian rhythms generated by an endogenous biologic clock and do not appear to be driven by the pattern of melatonin in circulation.

Long-Term Hypoxia Alters Endocrine and Physiologic Responses to Umbilical Cord Occlusion in the Ovine Fetus

Objective: This study was designed to determine the effect of umbilical cord occlusion (UCO) on fetal endocrine responses in the long-term hypoxemic (LTH) ovine fetus. Methods: Pregnant ewes were maintained at high altitude (3820 m) from day 30 of gestation. Normoxic control and LTH fetuses were catheterized, and an inflatable occluder was placed on the umbilical cord at day 132 of gestation. In the LTH group, maternal oxygen tension was maintained at approximately 60 mmHg by nitrogen infusion through a maternal tracheal catheter. On day 137, two 5-minute UCOs were performed. On day 139, the study was repeated with a 10-minute UCO. Results: Basal adrenocorticotropic hormone (ACTH) levels and peak responses to the first 5-minute UCO were not different between control and LTH fetuses (17.6 ± 4.0 to 418.8 ± 41.3 in controls, 25.7 ± 4.0 to 530.0 ± 93.0 pg/mL in LTH fetuses). A similar pattern was observed during the second UCO. Basal cortisol levels were similar in both groups. In response to UCO, a significant increase in cortisol was observed in both groups, but peak concentrations in the LTH group were significantly higher than those in the control group (23.9 ± 4.8 versus 14.8 ± 2.9 mg/mL, respectively, P < .05). The second occlusion also increased cortisol concentrations, but no differences were observed between groups. After the 10-minute UCO, the ACTH and cortisol responses were similar to the first 5-minute occlusion, with higher cortisol levels in the LTH fetuses. Conclusion: Despite similar ACTH responses to UCO, the cortisol response was greater in the LTH fetuses than in normoxic controls. LTH appears to result in enhanced adrenal sensitivity to a secondary stressor or altered cortisol metabolism.

Long-term hypoxia enhances ACTH response to arginine vasopressin but not corticotropin-releasing hormone in the near-term ovine fetus

This study tested the hypothesis that long-term hypoxia (LTH) results in enhanced fetal corticotrope sensitivity to the ACTH secretagogues, corticotropin-releasing hormone (CRH), and AVP. Ewes were maintained at high altitude (3,820 m) from 40 to 130-131 days of gestation. Upon return to the laboratory, hypoxia was maintained by maternal nitrogen infusion. Vascular catheters were placed in both LTH (n = 4) and normoxic controls (n = 4). Each fetus received a 15-min infusion of either saline, 100 ng/kg of ovine CRH, or 20 ng/kg of AVP/min over 3 consecutive days in a randomized order. Fetal blood samples were collected at 0, 15, 30, 60, and 90 min after the start of infusion and analyzed for ACTH(1-39), ACTH precursors, and cortisol. Anterior pituitaries were collected from additional noninstrumented fetuses for analysis of vasopressin receptor 1b (V1b) mRNA and protein. Basal plasma concentrations of both ACTH(1-39) and ACTH precursors were higher in LTH fetuses and were not altered by saline infusion. In response to CRH, ACTH(1-39) increased in both groups and was higher in the LTH group compared with control (P < 0.05). When analyzed as sum of ACTH(1-39) released (Delta0-90 min) above basal, CRH released equal amounts of ACTH(1-39) in both groups. In LTH fetuses, AVP evoked a greater ACTH(1-39) release (P < 0.05) when analyzed as an increased sum of ACTH(1-39) (Delta0-90 min) above basal. Both CRH and AVP elicited a release of ACTH precursors with no differences observed between LTH and control. AVP and CRH elicited significant increases in cortisol, which were higher in response to AVP than CRH. V1b mRNA and protein were elevated in the anterior pituitary of LTH fetuses compared with control. LTH significantly increases pituitary sensitivity to AVP. This enhanced sensitivity may be a mechanism of our previously observed enhanced corticotrope function.

Leptin receptor antagonist treatment ameliorates the effects of long-term maternal hypoxia on adrenal expression of key steroidogenic genes in the ovine fetus

We previously reported elevated adipose leptin expression, plasma leptin concentrations, and adrenocortical leptin receptor expression in the long-term hypoxic (LTH) ovine fetus. This study addressed whether leptin antagonist (LA) administration to LTH fetal sheep altered expression of key genes governing cortisol synthesis. Ewes were maintained at high altitude (3,820 meters) from 40 to 130 days gestation (dG), returned to Loma Linda University, and implanted with a maternal tracheal catheter. Reduced Po2 was maintained by nitrogen infusion. On 132 dG, LTH (n = 11) and age-matched, normoxic control (n = 11) fetuses underwent vascular catheter implantation. At 138 dG, fetuses were continuously infused with either saline or the LA (1.5 mg·kg(-1)·day(-1)) for 4 days and samples collected for blood gases, ACTH, and cortisol. Fetal adrenal cortex was collected for determination of steriodogenic acute regulatory protein (StAR), ACTH, and leptin receptor, cholesterol side-chain cleavage (CYP11A1), cytochrome P-450 11β-hydroxylase (CYP11B1), 17α-hydroxylase (CYP17), 21-hydroxylase (CYP21), signal transducer and activator of transcription 3 (STAT3), pSTAT3, and 17β-hydroxysteroid dehydrogenase (HSD3B) expression. In the saline-infused LTH fetuses, StAR, ACTH receptor, CYP11A1, and CYP17 expression was significantly lower compared with control (P < 0.05), whereas levels of CYP11B1, CYP21, and HSD3B mRNA were similar between groups. LA infusion restored expression of StAR, pSTAT3, CYP11A1, and CYP17, but not ACTH receptor, to normal ontogenic levels in the LTH group while having no effect on control fetuses. Neither fetal plasma ACTH nor cortisol concentrations were altered by LA infusion. We speculate that while leptin plays a role in governing expression of key enzymes and StAR in response to LTH, other factors play a role in modulating cortisol synthesis in these fetuses.

Long-Term Hypoxia Changes Myometrial Responsiveness and Oxytocin Receptors in the Pregnant Ewe: Differential Effects on Longitudinal Versus Circular Smooth Muscle

Abstract Previous studies from our laboratory demonstrated that long-term hypoxia (LTH) altered in vitro contractile responses to oxytocin in full-thickness myometrial strips from pregnant sheep. The present study was designed to determine, first, if the reduced contractile response to oxytocin following LTH is the result of combined effects on longitudinal and circular smooth muscle or if the effect is specific to a single muscle layer and, second, if the reduced contractile response to oxytocin following LTH is caused by changes in oxytocin-receptor protein. Pregnant ewes were maintained at high altitude (3820 m) from Day 30 to Days 137–142 of gestation, when the ewes were killed for collection of myometrial tissue. Tissue was also collected from age-matched, normoxic controls. Longitudinal and circular layers were separated, length-tension curves generated to determine optimal resting tension, and all strips exposed to increasing half-log doses of oxytocin ranging from 10–12 to 10–6.5 M. The expression of oxytocin-receptor protein was measured using Western blot analysis. We found that LTH did not affect KCl-induced contraction of either smooth muscle layer, whereas the sensitivity of both myometrial layers to oxytocin was altered. A decreased maximum contractile response of the circular layer to oxytocin was also observed. Additionally, LTH decreased expression of oxytocin-receptor protein in the circular layer and increased levels in the longitudinal layer. Results from the present study indicate that LTH alters contractile responses and oxytocin-receptor protein expression in a layer-specific manner in the pregnant sheep myometrium.

Long-term hypoxia enhances cortisol biosynthesis in near-term ovine fetal adrenal cortical cells.

This study was designed to determine the potential mechanism/mechanisms of previously observed enhanced fetal cortisol secretion following exposure to long-term hypoxia (LTH). Pregnant ewes were maintained at high altitude (3820 m) for approximately the last 100 days of gestation. Between the gestation days of 138 and 141, adrenal glands were collected from LTH and age-matched normoxic control fetuses. Cyclic adenosine monophosphate (cAMP), cortisol, and steroidogenic acute regulatory (StAR) protein were measured in response to adrenocorticotropic hormone (ACTH) stimulation. Cortisol responses to ACTH were also measured in the presence of the protein kinase (PKA) inhibitor H-89, proopiomelanocortin (POMC), or 22-kDa pro-ACTH. Cortisol output was higher in the LTH group compared to the control (P < .05), following ACTH treatment while the cAMP response was similar in both groups. Although PKA inhibition decreased cortisol production in both groups, however no differences were observed between groups. Western analysis revealed a significant increase in protein expression for StAR in the LTH group (P < .05, compared to control). Proopiomelanocortin and 22-kDa pro-ACTH did not alter the cortisol response to ACTH treatment. Results from the present study taken together with those of previous in vivo studies suggest that the enhanced cortisol output in the LTH group is not the result of differences in cAMP generation or PKA. We conclude that enhanced cortisol production in LTH adrenals is the result of enhanced protein expression of StAR and potential downstream signaling pathways.

Long-Term Gestational Hypoxia Modulates Expression of Key Genes Governing Mitochondrial Function in the Perirenal Adipose of the Late Gestation Sheep Fetus

We previously reported that long-term hypoxia (LTH) increases expression of brown adipose tissue (BAT) genes in the perirenal adipose in the ovine fetus. The mechanisms with which hypoxia mediates the enhanced BAT phenotype are unresolved. This study was designed to examine the effects of LTH on (1) the expression of endothelial cell nitric oxide synthase (eNOS) and (2) indicators of mitochondrial biogenesis (transcription factors mitochondrial transcription factor A (mtTFA), nuclear respiratory factor (NRF) 1, and NRF-2; cytochrome c oxidase (COX) I, II, and IV and mitochondrial DNA content). Pregnant ewes were maintained at high altitude (3820 m) from ∼40 to 137 to 140 days of gestation and perirenal adipose was collected from normoxic control and LTH fetuses. There was no effect of LTH on fetal body weight or perirenal adipose mass. Long-term hypoxia increased (P < .05) perirenal eNOS and phospho-eNOS, messenger RNA (mRNA) for NRF1, NRF-2, mtTFA as well as COX-I, COX-II, and COX-IV mRNA. In contrast, mRNA for 2 markers for cellular proliferation (Ki67 and proliferating cell nuclear antigen [PCNA]) was lower in perirenal adipose from LTH fetuses compared to controls (P < .05), while mitochondrial to nuclear DNA ratio did not differ between groups. In conclusion, nitric oxide may function as a mechanism via which LTH enhances the BAT phenotype in fetal sheep prior to birth. Although there is an apparent increase in genes supporting mitochondrial function and adaptive thermogenesis in response to LTH, there does not appear to be an increased mitochondrial biogenesis per se. Such adaptive changes may provide a mechanism for the prominence of the BAT phenotype observed in the late gestation LTH fetus.

Adrenocorticotropic Hormone and PI3K/Akt Inhibition Reduce eNOS Phosphorylation and Increase Cortisol Biosynthesis in Long-Term Hypoxic Ovine Fetal Adrenal Cortical Cells

This study was designed to determine the role of the MEK/ERK1/2 and PI3K/Akt pathways in cortisol production and endothelial nitric oxide synthase (eNOS) phosphorylation (peNOS) in the ovine fetal adrenal in response to long-term hypoxia (LTH). Pregnant ewes were maintained at high altitude (3820 m) for the last 100 days of gestation (dGa). At 138 to 142 dGa, fetal adrenal cortical cells (FACs) were collected from LTH and age-matched normoxic fetuses. Cortisol production and peNOS were measured in response to pretreatment with the MEK/ERK1/2 pathway inhibitor UO126 (UO) and adrenocorticotropic hormone (ACTH) stimulation. UO126 reduced ACTH-stimulated cortisol in both normoxic and LTH FACs. UO126 alone or in combination with ACTH reduced peNOS in the normoxic group, while ACTH alone or ACTH + UO inhibited peNOS in LTH FACs. Additionally, cortisol was measured in response to pretreatment with UO and treatment with 22R-hydroxycholesterol (22R-OHC) or water-soluble cholesterol (WSC) with and without ACTH stimulation. UO126 had no effect on 22R-OHC–treated cells, but reduced cortisol in cells treated with WSC and/or ACTH. Cortisol and peNOS were also measured in response to pretreatment with PI3K/Akt pathway inhibitor Wortmannin (WT) and ACTH stimulation. Wortmannin further increased cortisol under ACTH-stimulated conditions and, like ACTH, reduced peNOS in LTH but not normoxic FACs. Together, these data suggest that in LTH FACs MEK/ERK1/2 does not regulate peNOS but that UO acts downstream from eNOS, possibly at cholesterol transport, to affect cortisol production in LTH FACs, while the PI3K/Akt pathway, along with ACTH, regulates peNOS and plays a role in the fetal adaptation to LTH in FACs.

Gestational hypoxia modulates expression of corticotropin‐releasing hormone and arginine vasopressin in the paraventricular nucleus in the ovine fetus

Maturation of the fetal hypothalamo–pituitary–adrenocortical (HPA) axis is critical for organ maturation necessary for the fetus to transition to the ex‐utero environment. Intrauterine stressors can hasten maturation of the HPA axis leading to fetal growth restriction and in sheep, premature birth. We have previously reported that high‐altitude mediated, long‐term‐moderate gestational hypoxia (LTH) during gestation has a significant impact on the fetal HPA axis. Significant effects were observed at the level of both the anterior pituitary and adrenal cortex resulting in elevated plasma ACTH during late gestation with decreased adrenocortical expression of enzymes rate limiting for cortisol synthesis. As such, these fetuses exhibited the normal ontogenic rise in fetal plasma cortisol but an exaggerated cortisol response to acute stress. This study extended these findings to ACTH secretagogue expression in the PVN using in situ hybridization. We report that the expression of AVP but not CRH was increased in the medial parvocellular PVN (mpPVN) in the LTH fetus. This represented an increase in both AVP mRNA per neuron as well as an increase in AVP hybridizing neurons with no increase in mpPVN CRH neurons. LTH had no effect on PVN volume, area of CRH or AVP hybridization, thus LTH did not have a trophic effect on the size of the nucleus. In conclusion, there appears to be a switch from CRH to AVP as a primary ACTH secretagogue in response to LTH, supporting our previous findings of increased anterior pituitary sensitivity to AVP over CRH in the LTH fetus.