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1.
Reprod Sci ; 25(2): 230-238, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28468567

RESUMO

We previously demonstrated decreased expression of key genes regulating cortisol biosynthesis in long-term hypoxic (LTH) sheep fetal adrenals compared to controls. We also showed that inhibition of the extracellular signal-regulated kinases (ERKs) with the mitogen-activated protein kinase (MEK)/ERK inhibitor UO126 limited adrenocorticotropic (ACTH)-induced cortisol production in ovine fetal adrenocortical cells (FACs), suggesting a role for ERKs in cortisol synthesis. This study was designed to determine whether the previously observed decrease in LTH cytochrome P45011A1/cytochrome P450c17 (CYP11A1/CYP17) in adrenal glands was maintained in vitro, and whether ACTH alone with or without UO126 treatment had altered the expression of CYP11A1, CYP17, and steroidogenic acute regulatory protein (StAR) in control versus LTH FACs. Ewes were maintained at high altitude (3820 m) from ∼40 days of gestation (dG). At 138 to 141 dG, fetal adrenal glands were collected from LTH (n = 5) and age-matched normoxic controls (n = 6). Fetal adrenocortical cells were challenged with ACTH (10-8 M) with or without UO126 (10 µM) for 18 hours. Media samples were collected for cortisol analysis and messenger RNA (mRNA) for CYP11A1, CYP17, and StAR was quantified by quantitative real-time polymerase chain reaction. Cortisol was higher in the LTH versus control ( P < .05). StAR mRNA was decreased in LTH versus control ( P < .05). U0126 alone had no effect on mRNA in either group. UO126 prevented the increase in CYP11A1 and CYP17 in control FACs. Basal CYP11A1 and CYP17 were not different in LTH versus control. ACTH increased CYP11A1 and CYP17 only in control FACs ( P < .05). U1026 attenuated the ACTH response indicative of a role for ERK in CYP11A1 and CYP17 expression. ACTH may require additional factors in FACs to fully regulate StAR expression.


Assuntos
Córtex Suprarrenal/metabolismo , Hidrocortisona/metabolismo , Hipóxia/metabolismo , Fosfoproteínas/metabolismo , Córtex Suprarrenal/citologia , Córtex Suprarrenal/efeitos dos fármacos , Hormônio Adrenocorticotrópico/farmacologia , Animais , Butadienos/farmacologia , Sobrevivência Celular , Inibidores Enzimáticos/farmacologia , Hipóxia/genética , Nitrilas/farmacologia , Fosfoproteínas/genética , Ovinos
2.
PLoS One ; 12(9): e0185272, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28957383

RESUMO

The effect of gestational hypoxia on the neonatal leptin surge, development of hypothalamic arcuate nuclei (ARH) projections and appetite that could contribute to the programming of offspring obesity is lacking. We examined the effect of 12% O2 from gestational days 15-19 in the Sprague-Dawley rat on post-weaning appetite, fat deposition by MRI, adipose tissue cytokine expression, the neonatal leptin surge, ARH response to exogenous leptin, and αMSH projections to the paraventricular nucleus (PVN) in response to a high fat (HFD) or control diet (CD) in male offspring. Normoxia (NMX) and Hypoxia (HPX) offspring exhibited increased food intake when fed a HFD from 5-8 weeks post-birth; HPX offspring on the CD had increased food intake from weeks 5-7 vs. NMX offspring on a CD. HPX offspring on a HFD remained hyperphagic through 23 weeks. Body weight were the same between offspring from HPX vs. NMX dams from 4-12 weeks of age fed a CD or HFD. By 14-23 weeks of age, HPX offspring fed the CD or HFD as well as male NMX offspring fed the HFD were heavier vs. NMX offspring fed the CD. HPX offspring fed a CD exhibited increased abdominal adiposity (MRI) that was amplified by a HFD. HPX offspring fed a HFD exhibited the highest abdominal fat cytokine expression. HPX male offspring had higher plasma leptin from postnatal day (PN) 6 through 14 vs. NMX pups. HPX offspring exhibited increased basal c-Fos labeled cells in the ARH vs. NMX pups on PN16. Leptin increased c-Fos staining in the ARH in NMX but not HPX offspring at PN16. HPX offspring had fewer αMSH fibers in the PVN vs. NMX offspring on PN16. In conclusion, gestational hypoxia impacts the developing ARH resulting in hyperphagia contributing to adult obesity on a control diet and exacerbated by a HFD.


Assuntos
Hiperfagia/sangue , Hiperfagia/complicações , Hipóxia/sangue , Hipóxia/complicações , Leptina/sangue , Obesidade/sangue , Obesidade/complicações , Tecido Adiposo/metabolismo , Animais , Animais Recém-Nascidos , Ansiedade/sangue , Ansiedade/complicações , Núcleo Arqueado do Hipotálamo/metabolismo , Comportamento Animal , Dieta , Medo , Comportamento Alimentar , Feminino , Peso Fetal , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Imageamento por Ressonância Magnética , Masculino , Fenômenos Fisiológicos da Nutrição Materna , Aprendizagem em Labirinto , Atividade Motora , Gravidez , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Água , Desmame , alfa-MSH/metabolismo
4.
Biol Reprod ; 96(4): 866-876, 2017 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-28339937

RESUMO

Leptin regulates body weight, reproductive functions, blood pressure, endothelial function, and fetoplacental angiogenesis. Compared to the luteal phase, the follicular phase and pregnancy are physiological states of elevated estrogen, angiogenesis, and uterine blood flow (UBF). Little is known concerning regulation of uterine artery (UA) angiogenesis by leptin and its receptors. We hypothesized that (1) ex vivo expression of leptin receptors (LEPR) in UA endothelium (UAendo) and UA vascular smooth muscle (UAvsm) is elevated in pregnant versus nonpregnant (Luteal and Follicular) sheep; (2) in vitro leptin treatments differentially modulate mitogenesis in uterine artery endothelial cells from pregnant (P-UAECs) more than in nonpregnant (NP-UAECs) ewes; and (3) LEPR are upregulated in P-UAECs versus NP-UAECs in association with leptin activation of phospho-STAT3 signaling. Local UA adaptations were evaluated using a unilateral pregnant sheep model where prebreeding uterine horn isolation (nongravid) restricted gravidity to one horn. Immunolocalization revealed LEPR in UAendo and UAvsm from pregnant and nonpregnant sheep. Contrary to our hypothesis, western analysis revealed that follicular UAendo and UAvsm LEPR were greater than luteal, nongravid, gravid, and control pregnant. Compared to pregnant groups, LEPR were elevated in renal artery endothelium of follicular and luteal sheep. Leptin treatment significantly increased mitogenesis in follicular phase NP-UAECs and P-UAECs, but not luteal phase NP-UAECs. Although UAEC expression of LEPR was similar between groups, leptin treatment only activated phospho-STAT3 in follicular NP-UAECs and P-UAECs. Thus, leptin may play an angiogenic role particularly in preparation for the increased UBF during the periovulatory period and subsequently to meet the demands of the growing fetus.


Assuntos
Células Endoteliais/metabolismo , Ciclo Menstrual/fisiologia , Neovascularização Fisiológica/fisiologia , Receptores para Leptina/metabolismo , Ovinos/fisiologia , Artéria Uterina/metabolismo , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica/fisiologia , Gravidez , Receptores para Leptina/genética
5.
Am J Physiol Regul Integr Comp Physiol ; 304(8): R636-43, 2013 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-23427082

RESUMO

This study assessed the role of the extracellular signal-regulated kinase (ERK) signaling pathway on the previously observed enhanced cortisol secretion in response to adrenocorticotropic hormone (ACTH) treatment in fetal adrenocortical cells (FACs) from long-term hypoxic (LTH) ovine fetuses. Ewes were maintained at high altitude (3,820 m) from ~40 to 138-141 days gestation when FACs were collected and challenged with either ACTH (10 nM) or 8-bromoadenosine 3',5'-cyclic monophosphate (8-bromo-cAMP, 10 mM) in the presence or absence of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MEK)/ERK inhibitor UO126 (10 µM). FACs from age-matched normoxic fetuses served as controls. Media and FACs were collected at selected time intervals after ACTH or 8-bromo-cAMP stimulation for cortisol measurement and Western analysis of ERK1/2 and phospho-ERK1 and -2 (pERK1/2). After ACTH or 8-bromo-cAMP treatment, cortisol production was greater in the LTH group compared with control (P < 0.05). UO126 reduced ACTH and 8-bromo-cAMP-mediated cortisol output in both groups (P < 0.01 vs. ACTH or 8-bromo-cAMP alone). Under basal conditions, ERK1/2 and pERK1/2 were not different between LTH and normoxic fetuses. In response to ACTH or 8-bromo-cAMP treatment, ERK1/2 were not different between groups; however, pERK1/2 were elevated in the LTH FACs compared with normoxic control FACs. ERK1/2 phosphorylation declined following ACTH treatment in the control group, but UO126 had no effect on ERK1/2 compared with untreated levels. Both ACTH and 8-bromo-cAMP treatment resulted in a decline of protein levels. UO126 pretreatment virtually eliminated pERK1/2 expression. We conclude that basal ERK signaling in FACs is necessary for normal cortisol production and sustained pERK in LTH adrenals enhances cortisol production.


Assuntos
Glândulas Suprarrenais/embriologia , Glândulas Suprarrenais/metabolismo , Hipóxia Fetal/metabolismo , Hipóxia Fetal/fisiopatologia , Hidrocortisona/biossíntese , Sistema de Sinalização das MAP Quinases/fisiologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Córtex Suprarrenal/citologia , Córtex Suprarrenal/embriologia , Córtex Suprarrenal/metabolismo , Glândulas Suprarrenais/efeitos dos fármacos , Hormônio Adrenocorticotrópico/farmacologia , Animais , Western Blotting , Butadienos/farmacologia , Inibidores Enzimáticos/farmacologia , Feminino , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Gravidez , Ovinos
6.
Am J Physiol Regul Integr Comp Physiol ; 304(6): R435-42, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23344230

RESUMO

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 11ß-hydroxylase (CYP11B1), 17α-hydroxylase (CYP17), 21-hydroxylase (CYP21), signal transducer and activator of transcription 3 (STAT3), pSTAT3, and 17ß-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.


Assuntos
Córtex Suprarrenal/metabolismo , Feto/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hidrocortisona/biossíntese , Hipóxia/genética , Receptores para Leptina/antagonistas & inibidores , 17-Hidroxiesteroide Desidrogenases/metabolismo , Córtex Suprarrenal/efeitos dos fármacos , Animais , Feminino , Idade Gestacional , Hipóxia/metabolismo , Leptina/metabolismo , Receptores da Corticotropina/metabolismo , Ovinos , Esteroide 21-Hidroxilase/metabolismo , Fatores de Tempo
7.
Reprod Sci ; 18(3): 277-85, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21079237

RESUMO

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.


Assuntos
Córtex Suprarrenal/metabolismo , Hipóxia Fetal/metabolismo , Feto/metabolismo , Hidrocortisona/biossíntese , Ovinos/metabolismo , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Hidrocortisona/metabolismo , Fosfoproteínas/metabolismo , Gravidez
8.
Reprod Sci ; 17(10): 955-62, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20713972

RESUMO

We previously reported that in the sheep fetus, long-term hypoxia (LTH) resulted in elevated basal plasma adrenocorticotropic hormone (ACTH(1- 39)) whereas the cortisol levels were not different from normoxic controls. We also showed that LTH enhances endothelial nitric oxide synthase (eNOS) expression in the fetal adrenal. This study was designed to determine the effect of NO on cortisol production in adrenocortical cells from LTH fetal sheep. Ewes were maintained at high altitude (3820 m) from ∼40 days' gestation (dG) to near term. Between 138 and 141 dG, fetal adrenal glands were collected from LTH and age-matched normoxic control fetuses. Adrenal cortical cells were pretreated with sodium nitroprusside (SNP), nitro-L-arginine methyl ester (L-NAME), L-arginine, or diethyleneamine NO (DETA-NO) and then challenged with 10 nmol/L ACTH. Cortisol responses were compared after 1 hour. Adrenocorticotropic hormone -induced cortisol secretion was significantly higher in LTH versus control (P < .01). Enhancement of NO with L-arginine resulted in a significant reduction of ACTH-mediated cortisol production in the LTH group. DETA-NO also caused a significant decrease in ACTH-mediated cortisol production (P < .05). Inhibition of NOS with L-NAME significantly increased cortisol production in the LTH group (P < .05 compared to ACTH alone), whereas the effect on the control group was not significant. Nitric oxide synthase activity was significantly higher in the LTH group compared to control, but this difference was eliminated following ACTH treatment. These data indicate that LTH enhances adrenal cortical sensitivity to the inhibitory effects of NO on cortisol production. Nitric oxide may, therefore, play an important role in regulating ACTH-induced cortisol production in the LTH fetal adrenal.


Assuntos
Córtex Suprarrenal/metabolismo , Hormônio Adrenocorticotrópico/metabolismo , Hipóxia Fetal/metabolismo , Hidrocortisona/metabolismo , Óxido Nítrico/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Feto , Hidrocortisona/análise , NG-Nitroarginina Metil Éster/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Nitroprussiato/farmacologia , Gravidez , Ovinos , Triazenos/farmacologia
9.
Reprod Sci ; 16(9): 865-74, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19525401

RESUMO

This study was designed to test the hypothesis that fetal adrenal nitric oxide synthase (NOS) is elevated in response to long-term hypoxia (LTH). Pregnant ewes were maintained at high altitude (3820 m) for approximately the last 100 days of gestation. Between days 138 and 141 of gestation, adrenal glands were collected from LTH fetuses and age-matched normoxic controls. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western analysis were used to quantify NOS expression, and NOS distribution was examined by immunohistochemistry and double-staining immunofluorescence for endothelial NOS (eNOS) and 17alpha-hydroxylase (CYP17). Neuronal NOS (nNOS) was expressed at very low levels and with no differences between groups. Expression of eNOS was significantly greater in the LTH group compared with control. Neuronal NOS was distributed throughout the cortex while the greatest density of eNOS was observed in the zona fasciculata/reticularis area and eNOS co-localized with CYP17. We conclude that LTH enhances eNOS expression in the inner adrenal cortex which may play a role in regulation of cortisol biosynthesis in the LTH fetus.


Assuntos
Córtex Suprarrenal/enzimologia , Altitude , Hipóxia/fisiopatologia , Óxido Nítrico Sintase Tipo III/genética , Animais , Western Blotting , Doença Crônica , Feminino , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Hidrocortisona/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ovinos , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismo
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