The Effect of a Novel Glucocorticoid Receptor Antagonist (CORT113176) on Glucocorticoid and Insulin Receptor Sensitive Hepatic Gene (mRNA) Expression in a Neonatal Rat Model of Human Prematurity

Abstract Preterm birth is a global health problem the sequelae of which are not well understood. Hypoxia, a common stressor with prematurity, can affect blood glucose via stress-induced increases in glucocorticoids (GC). GCs are also administered to preterm infants to improve oxygenation; however, this is controversial. CORT113176 (Corcept Therapeutics) is a novel, selective glucocorticoid receptor (GR) antagonist that does not bind to the progesterone receptor. We have demonstrated that CORT113176 (in our rat model of preterm birth) increases baseline corticosterone (due to loss of GC negative feedback) and attenuates hypoxia-induced increases in insulin resistance implicating endogenous corticosterone in post-natal metabolic adaptations to stress. We now propose that CORT113176 is useful to evaluate the hepatic effects of endogenous GCs in our rat model of preterm birth by measuring critical GC and insulin receptor sensitive gene mRNAs. Postnatal day (PD) 2 rat pups of both sexes (N=5 per treatment/sex) were pretreated with CORT113176 (600 mg/kg IP) or vehicle. After 60 minutes, a group of pups were euthanized with livers collected and preserved in RNAlater (baseline). The remaining pups were separated from their dams, exposed to normoxia (control) or hypoxia (8% O2) for 60 minutes, and livers obtained. Total hepatic RNA was extracted, and mRNA expression was analyzed (RT-qPCR) for GC and insulin receptor sensitive genes: GC: Fkbp5, Gilz, Nr3c1 (Gr), Nr3c2 (Mr), Per1, Ttpa. INSULIN: Akt2, G6Pase, Igf1r, Insr, Irs1, Irs2, Pik3cb, Pik3r1, Srebp1c. CORT113176 decreased the expression of all baseline hepatic insulin receptor mRNAs in both sexes, except for G6Pase. Pik3r1 mRNA expression significantly decreased with 60 minutes of normoxic separation (fasting) in males and females compared to baseline and hypoxic separation; this was blocked by CORT113176. In the GC receptor sensitive panel, CORT113176 decreased basal Nr3c1 (Gr) mRNA. Normoxic and hypoxic separation increased Per1 and Gilz mRNA expression; this effect was blocked by CORT113176. Interestingly, Fkbp5 expression, a proposed clinical marker for GR antagonism, was not altered by CORT113176. The hepatic GC and insulin receptor sensitive gene mRNA panels we developed are sensitive to GR antagonism suggesting they may be a useful addition to Fkbp5. The increase in endogenous corticosterone, acting via GR, is critical in the hepatic response to stress in our neonatal rat model of hypoxia and prematurity.

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Glucocorticoid receptor antagonist alters corticosterone and receptor sensitive mRNAs in the hypoxic neonatal rat

Endocrinology ◽

10.1210/endocr/bqab232 ◽

2021 ◽

Author(s):

Ashley L Gehrand ◽

Jonathan Phillips ◽

Kyle D Welhouse ◽

Hana Siddiqui ◽

Matthew Schulgit ◽

...

Keyword(s):

Insulin Resistance ◽

Glucocorticoid Receptor ◽

Insulin Receptor ◽

Plasma Corticosterone ◽

Neonatal Rat ◽

Target Tissue ◽

Endogenous Hormones ◽

Rat Pups ◽

Pancreas Function ◽

Neonatal Rat Model

Abstract Hypoxia, a common stressor with preterm birth, increases morbidity and mortality associated with prematurity. Glucocorticoids (GC) are administered to the preterm infant to improve oxygenation; prolonged use of GCs remains controversial. We evaluated a selective glucocorticoid receptor (GR) antagonist (CORT113176) in our neonatal rat model of human prematurity to assess how fasting and hypoxia-induced increases in neonatal corticosterone affects endogenous hormones and endocrine pancreas function. Neonatal rat pups at postnatal day (PD) 2, PD8, and PD15 were pretreated with CORT113176 and, after 60 min of separation and fasting, exposed to hypoxia (8% O2) or control (normoxia) for 30 or 60 minutes while fasting was continued. Plasma corticosterone, ACTH, glucose, and insulin were measured and fasting HOMA-IR (index of insulin resistance) calculated. Glucocorticoid and insulin receptor sensitive gene mRNAs were analyzed in liver, muscle, and adipose to evaluate target tissue biomarkers. CORT113176 pretreatment augmented baseline and hypoxia-induced increases in corticosterone and attenuated hypoxia-induced increases in insulin resistance at PD2. Normoxic and hypoxic stress increased the hepatic GR sensitive gene mRNAs, Gilz and Per1; this was eliminated by pretreatment with CORT113176. CORT113176 pretreatment decreased baseline insulin receptor sensitive gene mRNAs Akt2, Irs1, Pik3r1, and Srebp1c at PD2. We show that CORT113176 variably augments the stress-induced increases in corticosterone concentrations (attenuation of negative feedback) and that GR is critical for hepatic responses to stress in the hypoxic neonate. We also propose that measurement of Gilz and Per1 mRNA expression may be useful to evaluate the effectiveness of GR antagonism.

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Epidermal growth factor reduces the development of necrotizing enterocolitis in a neonatal rat model

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00196.2001 ◽

2002 ◽

Vol 282 (1) ◽

pp. G156-G164 ◽

Cited By ~ 178

Author(s):

Bohuslav Dvorak ◽

Melissa D. Halpern ◽

Hana Holubec ◽

Catherine S. Williams ◽

Debra L. McWilliam ◽

...

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Mrna Expression ◽

Necrotizing Enterocolitis ◽

Rat Model ◽

Transforming Growth Factor ◽

Gastrointestinal Disease ◽

Neonatal Rat ◽

Epidermal Growth ◽

Neonatal Rat Model

Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of prematurely born infants. Maternal milk plays an important protective role against NEC development and is the major source of epidermal growth factor (EGF) for neonates. The aim of this study was to examine the effect of orally administered EGF on the incidence of NEC in a neonatal rat model. Newborn rats were artificially fed either with growth factor-free rat milk substitute (RMS) or RMS supplemented with 500 ng/ml of EGF (RMS+EGF). Experimental NEC was induced by exposure to asphyxia and cold stress. Development of NEC was evaluated by gross and histological scoring of damage in the ileum. Ileal EGF receptor (EGF-R), EGF, and transforming growth factor-α mRNA expression was assessed by RT competitive-PCR, and the EGF-R was localized by immunohistochemistry. EGF supplementation of formula reduced the incidence and severity of NEC in rats (13/16 RMS vs. 4/13 RMS+EGF). Ileal EGF-R mRNA expression was markedly increased in the RMS group compared with RMS+EGF. Enhanced EGF-R expression in the RMS group was localized predominantly in the epithelial cells of injured ileum. These data suggest a new potential therapeutic approach for the prevention and treatment of NEC.

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Rotavirus Viremia and Extraintestinal Viral Infection in the Neonatal Rat Model

Journal of Virology ◽

10.1128/jvi.80.10.4820-4832.2006 ◽

2006 ◽

Vol 80 (10) ◽

pp. 4820-4832 ◽

Cited By ~ 94

Author(s):

Sue E. Crawford ◽

Dinesh G. Patel ◽

Elly Cheng ◽

Zuzana Berkova ◽

Joseph M. Hyser ◽

...

Keyword(s):

Rat Model ◽

Specific Antigen ◽

Portal Tract ◽

Neonatal Rat ◽

Enzyme Linked Immunosorbent Assay ◽

Rotavirus Infection ◽

Multiple Organs ◽

Rat Pups ◽

Residual Blood ◽

Neonatal Rat Model

ABSTRACT Rotaviruses infect mature, differentiated enterocytes of the small intestine and, by an unknown mechanism, escape the gastrointestinal tract and cause viremia. The neonatal rat model of rotavirus infection was used to determine the kinetics of viremia, spread, and pathology of rotavirus in extraintestinal organs. Five-day-old rat pups were inoculated intragastrically with an animal (RRV) or human (HAL1166) rotavirus or phosphate-buffered saline. Blood was collected from a subset of rat pups, and following perfusion to remove residual blood, organs were removed and homogenized to analyze rotavirus-specific antigen by enzyme-linked immunosorbent assay and infectious rotavirus by fluorescent focus assay or fixed in formalin for histology and immunohistochemistry. Viremia was detected following rotavirus infection with RRV and HAL1166. The RRV 50% antigenemia dose was 1.8 × 103 PFU, and the 50% diarrhea dose was 7.7 × 105 PFU, indicating that infection and viremia occurred in the absence of diarrhea and that detecting rotavirus antigen in the blood was a more sensitive measure of infection than diarrhea. Rotavirus antigens and infectious virus were detected in multiple organs (stomach, intestines, liver, lungs, spleen, kidneys, pancreas, thymus, and bladder). Histopathological changes due to rotavirus infection included acute inflammation of the portal tract and bile duct, microsteatosis, necrosis, and inflammatory cell infiltrates in the parenchymas of the liver and lungs. Colocalization of structural and nonstructural proteins with histopathology in the liver and lungs indicated that the histological changes observed were due to rotavirus infection and replication. Replicating rotavirus was also detected in macrophages in the lungs and blood vessels, indicating a possible mechanism of rotavirus dissemination. Extraintestinal infectious rotavirus, but not diarrhea, was observed in the presence of passively or actively acquired rotavirus-specific antibody. These findings alter the previously accepted concept of rotavirus pathogenesis to include not only gastroenteritis but also viremia, and they indicate that rotavirus could cause a broad array of systemic diseases in a number of different organs.

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Effects of Zidovudine Treatment on Heart mRNA Expression and Mitochondrial DNA Copy Number Associated with Alterations in Deoxynucleoside Triphosphate Composition in a Neonatal Rat Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01180-15 ◽

2015 ◽

Vol 59 (10) ◽

pp. 6328-6336 ◽

Cited By ~ 3

Author(s):

Jacob W. Snowdin ◽

Chia-Heng Hsiung ◽

Daniel G. Kesterson ◽

Vasudeva G. Kamath ◽

Edward E. McKee

Keyword(s):

Mitochondrial Dna ◽

Mrna Expression ◽

Rat Model ◽

Neonatal Rat ◽

Mrna Levels ◽

Hypertrophic Growth ◽

Hiv Therapy ◽

Mitochondrial Dna Copy Number ◽

Deoxynucleoside Triphosphate ◽

Neonatal Rat Model

ABSTRACTThe prevention of mother-to-child transmission (MTCT) of HIV is a crucial component in HIV therapy. Nucleoside reverse transcriptase inhibitors (NRTIs), primarily 3′-azido-3′-thymidine (AZT [zidovudine]), have been used to treat both mothers and neonates. While AZT is being replaced with less toxic drugs in treating mothers in MTCT prevention, it is still commonly used to treat neonates. Problems related to mitochondrial toxicity and potential mutagenesis associated with AZT treatment have been reported in treated cohorts. Yet little is known concerning the metabolism and potential toxicity of AZT on embryonic and neonatal tissues, especially considering that the enzymes of nucleoside metabolism change dramatically as many tissues convert from hyperplastic to hypertrophic growth during this period. AZT is known to inhibit thymidine phosphorylation and potentially alter deoxynucleoside triphosphate (dNTP) pools in adults. This study examines the effects of AZT on dNTP pools, mRNA expression of deoxynucleoside/deoxynucleotide metabolic enzymes, and mitochondrial DNA levels in a neonatal rat model. Results show that AZT treatment dramatically altered dNTP pools in the first 7 days of life after birth, which normalized to age-matched controls in the second and third weeks. Additionally, AZT treatment dramatically increased the mRNA levels of many enzymes involved in deoxynucleotide synthesis and mitochondrial biogenesis during the first week of life, which normalized to age-matched controls by the third week. These results were correlated with depletion of mitochondrial DNA noted in the second week. Taken together, results demonstrated that AZT treatment has a powerful effect on the deoxynucleotide synthesis pathways that may be associated with toxicity and mutagenesis.

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SUN-214 A Novel Glucocorticoid Receptor Antagonist (CORT113176) Reveals Unique Developmental and Tissue-Specific Effects in a Neonatal Rat Model of Human Prematurity

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.321 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Kyle D Welhouse ◽

Ashley Gehrand ◽

Jonathan Phillips ◽

Hana Siddiqui ◽

Matthew Schulgit ◽

...

Keyword(s):

Glucocorticoid Receptor ◽

Rat Model ◽

Premature Birth ◽

Neonatal Rat ◽

Whole Body ◽

Target Tissue ◽

Plasma Acth ◽

Corticosteroid Administration ◽

Tissue Specific ◽

Neonatal Rat Model

Abstract Premature birth is a major public health problem worldwide and can lead to transient adrenal insufficiency1,2. The stress of premature birth includes the inability to control blood glucose and maintain normal oxygenation leading to hypoxia3. Corticosteroid administration enhances surfactant production and improves oxygenation in preterm humans. However, corticosteroids can also have negative consequences4,5. We have validated a rat model of separation and hypoxia on post-natal day (PD) 2 that emulates the stress and treatment of hypoxia in the preterm human infant6,7. We hypothesized that the role of endogenous glucocorticoids in our neonatal rat model of preterm birth can be evaluated using the novel selective glucocorticoid receptor (GR) antagonist CORT113176 (Corcept) which is devoid of progesterone receptor effects8. Pups (PD 2, 8, or 15; N=6–8 per treatment/timepoint) were given CORT113176 (60 mg/kg IP) or vehicle, then placed into chambers in room air with mild warming to prevent hypothermia due to maternal separation. 60 min later, one group of pups was euthanized and trunk blood collected (baseline). The remaining pups were exposed to hypoxia (8% O2) or normoxia (time control) for 30 or 60 min at which times trunk blood was collected for the measurement of plasma glucose, insulin, ACTH, and corticosterone. Plasma ACTH, corticosterone, and insulin were measured by immunoassay. Glucose was measured by glucose oxidase method and insulin sensitivity calculated (HOMA-IR). Organs were frozen (brain, pituitary, adrenal glands, kidney, liver, muscle, fat) for future assessment of tissue-specific glucocorticoid-sensitive gene expression. In PD2 rats, basal and hypoxia-stimulated plasma ACTH and corticosterone were lower and basal HOMA-IR greater with CORT113176 pretreatment suggesting (unexpectedly) glucocorticoid agonist activity. In PD8 and PD15 rats, basal and hypoxia-stimulated plasma ACTH and corticosterone were augmented after CORT113176 pretreatment demonstrating classic antagonist activity. However, in PD8 rats, CORT113176 effects were tissue-specific acting as a classic antagonist on the HPA-axis, but as an agonist on whole-body insulin resistance. The differential effects of CORT113176 based on age and target tissue indicate that GR regulation changes in early development in our animal model of human prematurity. These findings may have significant implications in the treatment of hypoxia and transient adrenal insufficiency in the preterm infant1,2 as well as give insight into the nuances of the control of glucocorticoid receptor function. 1Lancet 392:1923–1994, 2018 2Curr Opin Endocrinol Diabetes Obes 17:8–12, 2010 3Compr Ther 13:14–19, 1987 4Int Immunopharmacol 66:242–250, 2019 5J Neuroendocrinol 27:468–480, 2015 6Am J Physiol 300:R708-715, 2011 7Am J Physiol 302:R627-R633, 2012 8Med Chem Lett 25:5720−5725, 2015

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