Adrenomedullin: new inhibitory regulator for cortisol synthesis and secretion
Simon TraversOD1,2, Laetitia Martinerie1,3,4,5, Qiong-Yao Xue1,2, Julie Perrot1, Say Viengchareun1, Kathleen M Caron6, Elizabeth S Blakeney6, Pascal Boileau1,7,8, Marc Lombès1,5 and Eric Pussard1,2
1Université Paris-Saclay, Inserm, Physiologie et Physiopathologie Endocriniennes, Le Kremlin-Bicêtre, France 2Service de Génétique Moléculaire, Pharmacogénétique et Hormonologie, Hôpital de Bicêtre, Assistance Publique-Hôpitaux de Paris, Le Kremlin Bicêtre, France
3Service d’Endocrinologie Pédiatrique, Hôpital Robert Debré, Assistance Publique Hôpitaux de Paris, Paris, France
4Université de Paris, Faculté de Santé, UFR de Médecine, Paris, France 5PremUp Fondation, Paris, France
6Department of Cell Biology and Physiology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, USA 7Department of Neonatal Pediatrics, Centre Hospitalier Intercommunal de Poissy-Saint-Germain, Poissy, France
8UFR des Sciences de la Santé, Simone Veil. Université Versailles St-Quentin en Yvelines, Montigny le Bretonneux, France
Correspondence should be addressed to S Travers: simon.travers@aphp.fr
Abstract
Preterm birth is associated with immaturity of several crucial physiological functions notably those prevailing in the lung and kidney. Recently, a steroid secretion deficiency was identified in very preterm neonates, associated with a partial yet transient deficiency in 116-hydroxylase activity, sustaining cortisol synthesis. However, the P450c110 enzyme is expressed in preterm adrenal glands, we hypothesized an inhibition of cortisol production by adrenomedullin (ADM), a peptide highly produced in neonates and whose effect on steroidogenesis remains poorly known. We studied the effects of ADM on three models: 104 cord-blood samples of the PREMALDO neonate cohort, genetically targeted mice overexpressing ADM, and two human adrenocortical cell lines (H295R and HAC15 cells). Mid-regional-proADM (MR-proADM) quantification in cord-blood samples showed strong negative correlation with gestational age (P = 0.0004), cortisol production (P < 0.0001), and 11ß-hydroxylase activity index (P < 0.0001). Mean MR-proADM was higher in very preterm than in term neonates (1.12 vs 0.60 nmol/L, P < 0.0001). ADM-overexpression mice revealed a lower 116-hydroxylase activity index (P < 0.05). Otherwise, aldosterone levels measured by LC-MS/MS were higher in ADM- overexpression mice (0.83 vs 0.46 ng/ml, P < 0.05). More importantly, the negative relationship between adrenal ADM expression and aldosterone production found in control was lacking in the ADM-overexpression mice. Finally, LC-MS/MS and gene expression studies on H295R and HAC15 cells revealed an ADM-induced inhibition of both cortisol secretion in cell supernatants and CYP11B1 expression. Collectively, our results converge toward an inhibitory effect of ADM on glucocorticoid synthesis in humans and should be considered to explain the steroid secretion deficiency observed at birth in premature newborns.
Key Words
adrenomedullin
adrenal
cortisol
hCYP11B1
prematurity
Journal of Endocrinology (2021) 251, 97-109
| Journal of Endocrinology | S Travers et al. | Adrenomedullin inhibits | 251:1 | 98 |
|---|---|---|---|---|
| cortisol secretion |
Introduction
Corticosteroid hormones are synthesized in the adrenal cortex, an endocrine gland that produces mineralocorticoids in the zona glomerulosa, glucocorticoids in the zona fasciculata, and adrenal androgens in the zona reticularis. Each zone is thereby dedicated to a specific steroidogenic pathway under specific regulatory mechanisms (Miller & Auchus 2011). Glucocorticoid production and secretion are mainly under the control of ACTH whereas mineralocorticoid synthesis responds to angiotensin II (Ang II) and potassium (K+) (Miller & Auchus 2011).
Prematurity is known to be associated with renal tubular immaturity that leads to poor electrolyte and water homeostasis, with major urinary sodium waste (Sulyok et al. 1979, Holtbäck & Aperia 2003). This transient physiological condition is linked to an inappropriate response to corticosteroid hormones at birth, as recently shown by the PREMALDO cohort (Martinerie et al. 2015).
An exhaustive steroidomic analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) revealed in this cohort a defect of both cortisol and aldosterone secretion in very preterm neonates. This defect was supported by a partial yet transient deficiency in 11-hydroxylase activity (P450c116, encoded by CYP11B1), at birth and at day 3 of life (Travers et al. 2017a), despite the fact that this enzyme is clearly expressed in adrenal glands at this developmental stage (Naccache et al. 2016). This latter enzymatic activity (P450c116), encoded by the CYP11B1 gene, is required to transform 11-deoxycortisol (S) into cortisol (F) (Schiffer et al. 2015). However, the expression of CYP11B1 has never been quantified according to the degree of prematurity. The reason for this partial deficiency remains unclear, but several hypotheses were made.
In order to elucidate the mechanisms of such a defect in steroidogenesis, we took a particular interest in the 52-amino acid peptide adrenomedullin (ADM), highly expressed in newborns (Marinoni et al. 1999, De Martin et al. 2014), whose plasma concentration is known to be increased during birth stress (Boldt et al. 1998) and used as a biomarker for brain injury in preterm infants (Gazzolo et al. 2001, Risso et al. 2012).
However, no study has assessed the action of ADM on cortisol synthesis, and the impact of ADM upon aldosterone production remains controversial. On one hand, ADM was shown to stimulate the renin-angiotensin- aldosterone system (RAAS) by increasing renin secretion through its vasodilatory effect (Jensen et al. 1997), to enhance steroid secretion ex vivo (Mazzocchi et al. 1996a)
and to increase aldosterone synthase (P450c11AS, encoded by CYP11B2) expression in H295R cells, a human adrenal cell line model (Thomson et al. 2001). On the other hand, ADM was reported to exert ex vivo and in vivo an inhibitory effect over aldosterone production in the rat (Yamaguchi et al. 1995, 1996, Mazzocchi et al. 1996b). These results are further complicated by the possibility of an autocrine and paracrine role of ADM produced in the human adrenal medulla (Thomson et al. 2001, 2003).
ADM production is mainly reported in the lung, kidney, placenta, adrenal medulla (hence its name), and cardiovascular system, as ADM is mainly synthesized and secreted by endothelial and vascular smooth muscle cells (Schönauer et al. 2017). The human ADM is encoded by a single gene located on chromosome 11 and transcripts lead to the prepro-ADM, a precursor converted into the pro-ADM that is then processed into four peptides: pro- ADM N-terminal 20 peptide, mid regional pro-ADM (MR-proADM) lacking any biological activity (Ishimitsu et al. 1994), immature ADM, and adrenotensin (Ishimitsu et al. 1994, Kitamura et al. 2002). Immature ADM is transformed into a bioactive peptide by amidation of its C-terminal part (Kitamura et al. 1998). ADM’s half-life is relatively short (22 min), whereas MR-proADM’s is several hours long (Valenzuela-Sánchez et al. 2016). As MR-proADM is secreted in equimolar quantity than ADM, MR-proADM is largely used as a biomarker of ADM production (Vigué et al. 2016, Gille et al. 2017, Krintus et al. 2018).
ADM effects are mediated through a multiprotein complex receptor consisting of the calcitonin-like receptor (CLR) and a co-receptor (receptor activity-modifying protein or RAMP) (Schönauer et al. 2017). Three types of RAMP have been described, and the association of RAMP2 or RAMP3 with CLR is known to have the highest specificity for ADM binding (McLatchie et al. 1998). Homozygous knockout mouse models for ADM are embryonic lethal (Caron & Smithies 2001), whereas an ADM-overexpressing mouse model presents with cardiac hyperplasia (Wetzel- Strong et al. 2014) improved lymphangiogenesis (Trincot et al. 2019), and revealed that ADM played a role in the innate immune milieu of the placenta (Li et al. 2013).
In this study, we hypothesized that the partial defect in steroid secretion observed during the first 3 days of human life, in very preterm neonates, was linked to the ADM signaling pathway. We measured MR-proADM in cord-blood samples from the PREMALDO cohort and performed in vitro studies to explore the underlying mechanisms that could lead to the partial defect in CYP11B1 expression in this fragile population. We also used an ADM-overexpressing mouse model to investigate
the potential impact of ADM in vivo and demonstrated that ADM inhibits cortisol production and secretion in two human H295R and HAC15 adrenocortical cell lines.
Materials and methods
Reagents
Forskolin (FK), adrenomedullin (ADM), and angiotensin II (Ang II) were obtained from Merck-Sigma-Aldrich. Another supplier for ADM was ThermoFisher but showed no pharmacological activity.
Newborn samples
MR-proADM quantification was performed on 104 cord- blood sample leftovers of the PREMALDO cohort already described (Martinerie et al. 2015). Newborns were sorted according to their gestational age: 30 very preterm (<33 gestational weeks (GW)), 43 preterms (33-36 GW), and 31 term neonates (≥37 GW). Precise gestational ages in days were available for 66 neonates. Steroidomic profiles were previously performed on these cord-blood samples and discussed elsewhere (Travers et al. 2017a).
ADM-overexpression mouse model
A mouse model overexpressing ADM (Admhi/hi) has been previously described. Briefly, the endogenous ADM mRNA was stabilized by replacing the 3’-UTR of Adm gene with that of the bovine growth hormone in a C57BL/6J genetic background, which allows approximately to triple the basal circulating ADM concentrations (Wetzel-Strong et al. 2014). The Admhi/hi mice are maintained on an isogenic C57BL/6J homozygous background breeding scheme. Therefore, Admhi/hi and C57BL/6J WT mice used in these studies were maintained and generated on isogenic genetic backgrounds. We studied seven WT male mice aged from 117 to 133 days, and six Admhi/hi male mice aged from 34 to 159 days, with blood samples and two adrenal glands from all mice. Animals were housed in specific pathogen- free housing with Tecniplast Greenline ventilated micro isolator cages. The mice had a 12 h light: 12 h darkness cycle from 07:00 on to 19:00 h off, with a temperature ranging between 68 and 74ºF (20-23℃) and humidity between 30 and 70%. Blood samples were collected via retro-orbital bleeds. Whole blood was collected in BD Microtainer tubes with BD SST additive. Whole blood was placed on ice for 30 min, then spun at 8000 g at 4℃. The supernatant
was collected, then frozen at -80℃ prior to shipping on dry ice. Mice were euthanized with carbon dioxide and secondary cervical dislocation before being dissected for adrenal glands.
All animal studies were approved by the Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill.
Cell culture
The human adrenocortical carcinoma H295R (RRID:CVCL_0458) and HAC15 (RRID:CVCL_S898) cells were tested and the latter were known for their higher steroidogenic capacities (Rainey et al. 2004). Cells were cultured in DMEM/HAM’S F12 medium (GE Healthcare) supplemented with 10% fetal calf serum (Biowest, Nuaillé, France), 20 mM HEPES (Life Technologies), penicillin (100IU/mL), streptomycin (100µg/mL), 2 mM glutamine, and 50 nM sodium selenite (Sigma), 5µg/mL transferrin (Life Technologies), and 5 µg/mL insulin (Sigma), at 37°℃ in a humidified atmosphere of 95% air and 5% CO2. HAC15 cells were cultured on type I collagen-coated plates (final solution concentration: 40 µg/mL, with 300 µL in each 12-well plates). Type I collagen was provided by Institut de Biotechnologies Jacques Boy (Reims, France).
For stimulation experiments, cells were seeded at a density of 150 × 103 cells/well. After 3 days of cell division with daily renew medium, cells were incubated in a complete medium supplemented with 10% dextran- coated charcoal serum and FK (20 µM), ADM (1 µM), Ang II (1 µM), potassium (K+, 10 mM), or the combination (FK+ADM) at described concentrations. Three hours stimulation was conducted for gene expression study, whereas 24 h stimulation was necessary for steroid secretion determination.
Hormone assays
Steroids levels in cell culture supernatants or in plasma/ serum samples were assessed by liquid chromatography coupled to tandem mass spectrometry as previously described (Travers et al. 2017b). Briefly, supernatants were collected from cell cultures and centrifuged at 9500 g for 5 min. Two hundred microliters of the supernatant were then extracted by the SPE method with deuterated internal standards and injected into the chromatographic system. Steroid hormones quantified are referred as follows: 11-deoxycorticosterone (DOC), corticosterone (B), 18-hydroxy-corticosterone (18OHB), aldosterone (Aldo), 11-deoxycortisol (S), and cortisol (F).
MR-proADM was assessed in cord-blood samples using TRACE (time-resolved amplified cryptate emission) technology, on a Kryptor compact PLUS® (ThermoFisher Scientific) (Caruhel et al. 2009).
Reverse transcription quantitative PCR (RT-qPCR)
RT-qPCR was performed as previously described (Viengchareun et al. 2009). Briefly, total RNAs were extracted using the Nucleospin RNAII® (Macherey Nagel, Hoerdt, France). After DNAse I treatment (Biolabs), total RNAs were reverse-transcribed using the High-Capacity CDNA RT Kit (Life Technologies). Samples were analyzed by quantitative PCR using the Power SYBR Green PCR Master Mix (Life Technologies) with the indicated primers (from Eurogentec, Liège, Belgium, Table 1) in a QuantStudio 6 Instrument (Life Technologies). For hCYP11B2 expression quantification, we used a TaqMan probe (Life Technologies) whose sequence was CGCCTTCAACACTAC.
Relative expression analysis was determined with a standard curve. For the preparation of standards, amplicons were subcloned into pGEMT-easy plasmid (Promega) and sequenced to confirm the identity of the sequence. Standard curves were generated using serial dilutions of linearized standard plasmids, spanning 6 orders of magnitude. Standard and sample values were amplified in duplicate and analyzed from two independent experiments. Results are presented as mean + S.D. and represent the relative expression of target genes normalized to 36b4 expression for mouse samples, and generally to geometric means of three housekeeping genes (ACTB, 36B4, GAPDH) for in vitro human cell samples, using the BestKeeper program (Pfaffl et al. 2004).
Statistical analyses
Results were analyzed on GraphPad Prism Software (v.8). Statistical tests are indicated in each figure legend, and statistical significance is as follow, according to P-value: *P<0.05; ** P<0.01; *** P<0.001; **** P <0.0001. Parametric correlations were performed using Pearson analysis, whereas non-parametric correlations were assessed using Spearman method.
Results
ADM quantification in PREMALDO cohort
We used the cord-blood leftovers of the PREMALDO cohort (5), composed of 30 samples from the very preterm (VTP)
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Printed in Great Britain
| Gene | Access number | Forward | Forward localization | Reverse | Reverse localization | Size (bp) |
|---|---|---|---|---|---|---|
| hCYP11B1 | NM_000497.4 | GGAGACACTAACCCAAGAGGACAT | Exon 9 | ACGTGATTAGTTGATGGCTCTGAA | Exon 9 | 100 |
| hCYP11B2 | NM_000498.3 | ATCTACCAGGAACTGGCCTTCA | Exon 5 | TGACAGTTCCGCCTTCAACA | Exon 5 | 81 |
| hADM | NM_001124.3 | CCGAGTGTTTGCCAGGCTTA | Exon 4 | GGTGACACGCCGTGAGAAAT | Exon 4 | 119 |
| hCLR | NM_005795.6 | AAGATTATGCAAGACCCCATTCA | Exon 5 | ATCAGGGCAGAGCTGCATTG | Exon 6 | 120 |
| hRAMP2 | NM_005854.3 | ACCAGATCCACTTTGCCAAC | Exon 4 | CCTGGGCCTCACTGTCTTTA | Exon 4 | 153 |
| hRAMP3 | NM_005856.3 | GCCATGGAGACTGGA | Exon 1 | CATCATGTCTGCGAAAG | Exon 2 | 146 |
| hACTB | NM_001101.5 | GCATGGGTCAGAAGGATTCCT | Exon 3 | ACACGCAGCTCATTGTAGAAGG | Exon 3 | 150 |
| h36B4 | NM_001002.4 | CCCATTCTATCATCAACGGGTACAA | Exon 7 | CAGCAAGTGGGAAGGTGTAATCC | Exon 7 | 75 |
| hGAPDH | NM_002046.7 | TGCACCACCAACTGCTTAGC | Exon 7 | GGCATGGACTGTGGTCATGAG | Exons 7 and 8 | 86 |
| mADM | NM_009627.2 | GAGCGAAGCCCACATTCGT | Exon 4 | GAAGCGGCATCCATTGCT | Exon 4 | 75 |
| mCYP11B1 | NM_001033229.3 | ACAAGCTGTAGACTTGTGGT | Exon 9 | GAAACGCACTAAACAATTTC | Exon 9 | 53 |
| mCYP11B2 | NM_009991.4 | TGAGTATGCCAACAGATGTA | Exon 4 & 5 | ACAATGCCACTGTAGGTCT | Exon 5 | 77 |
| m36B4 | NM_007475.5 | AGCGCGTCCTGGCATTGTCTGT | Exon 6 | GGGCAGCAGTGGTGGCAGCAGC | Exon 7 | 128 |
| Journal of | S Travers et al. | Adrenomedullin inhibits | 251:1 | 101 |
|---|---|---|---|---|
| Endocrinology | cortisol secretion |
group, 43 from the preterm (PT) group, and 31 from the term neonate group. We highlighted a decrease in cord- blood ADM as pregnancy progresses. Therefore, VPT secrete much more ADM at birth than term neonates (median and 25th-75th percentile): 1.11 nmol/L (0.62-1.61) vs 0.66 nmol/L (0.35-0.84), P <0.0001, as for PT whose ADM secretion is higher than term newborns (0.87 nmol/L (0.62-1.16), P < 0.05) (Fig. 1A). For 66 newborns whose gestational age was available in days, a negative correlation was observed between MR-proADM levels and gestational age (r =- 0.43, P=0.0004, Fig. 1B).
Moreover, studying the relationship between ADM and cortisol concentrations reported in Travers et al. (Travers et al. 2017a) for the PREMALDO cohort revealed a strong negative relationship (y =- 29.55x+55.52; r =- 0.53, P < 0.0001; Fig. 1C). In addition, the activity of the P450c110, estimated by the enzyme activity index (EAI) defined as a product-to-substrate ratio (F/S), was negatively correlated to MR-proADM levels (y =- 10.15x+20.33; r =- 0.40, P < 0.0001; Fig. 1D). On the contrary, no relationship was found between MR-proADM levels and aldosterone secretion (Fig. 1E).
It is very likely from these results that the more premature the birth is, the more ADM is secreted.
As preterm neonates are known to exhibit a steroid secretion deficiency at birth, we aimed at exploring the potential link between steroid production or secretion and ADM by studying steroid metabolomics in a genetically engineered mouse model overexpressing ADM, the Admhi/hi mouse model (Kitamura et al. 2002).
ADM overexpression in mouse is associated with loss of the inverse relation between aldosterone secretion and ADM levels observed in WT mice
We first quantified ADM expression by RT-qPCR in total adrenal glands (Fig. 2A) and showed, as expected, a ~2-fold higher ADM expression in Admhi/hi mice compared to WT controls (P < 0.01), consistent with prior characterization of the ADM-overexpression model.
Steroidomic profiling by LC-MS/MS showed no difference regarding corticosterone (B) secretion (Fig. 2B) but a lower corticosterone-to-11-deoxycorticosterone (B/DOC) ratio in Admhi/hi mice (P < 0.05, Fig. 2C). As B can be formed from DOC by P450c116 or P450cAS, this latter (B/DOC) ratio reflects activities from both enzymes (P450c116 and P450cAS). As 18OHB/B and Aldo/18OHB
A
MR-proADM
B
MR-proADM
2.5
2.53
*
MR-proADM (nmol/L)
r= - 0.43
2.0
2.0
nmol/L
P=0.0004
1.5
1.5
1.0
1.0
0.5
0.5
0.0
< 33 GW
33-36 GW
≥ 37 GW
0.0
180
200
220
240
260
280
300
Gestational age (days)
C
D
Cortisol
EAlp450c11₿ (F/S)
E
Aldosterone
150-
100g
2.02
Aldosterone (ng/ml)
r = - 0.53
80
100
P<0.0001
60
r= - 0.40
1.5
F (ng/ml)
F/S
P<0.0001
40
1.0
50
20
0.5
0
0.5
1.0
1.5
2.0
2.5
0
0.5
1.0
1.5
2.0
2.5
-20
0.0
0.0
0.5
1.0
1.5
2.0
2.5
-50-
MR-proADM
MR-proADM
MR-proADM
A
ADM expression
B
Corticosterone
Transcript expression relative to 36B4
300
ns
0.5
**
0.4
ng/ml
200
0.3
:
0.2
100
:
0.1
0
0.0
WT
Admhi/hi
WT
Admhi/hi
C
EAlP450c116 & P450c11AS (B/DOC)
D
Aldosterone
200
*
1.5
*
150
B/DOC
ng/mL
1.0
100
0.5
50
0
WT
Admhi/hi
0.0
WT
Admhi/hi
E
ADM vs Aldosterone
Aldosterone (ng/ml)
2.0-
- Aldo WT
- Admhi/hi
1.5
p=0.024
p=0.39 (ns)
1.0-
r = - 0.86
0.5-
0.0-
0.0
0.1
0.2
0.3
0.4
0.5
ADM expression
ratios that reflect P450cAS activity were unchanged (data not shown), the decreased B/DOC ratio could mainly be explained by P450c116 inhibition.
However, no difference in mCyp11b1 or mCyp11b2 expression was observed in adrenal glands of both groups, as determined by RT-qPCR (data not shown).
Surprisingly, steroid metabolomic studies allowed us to show an increase in serum aldosterone (0.83 ± 0.11 vs 0.46 ± 0.07 ng/ml, P < 0.05; mean ± S.E.M., Fig. 2D) and 18-hydroxy-corticosterone concentrations in Admhi/hi mice (data not shown), suggesting an increase in mineralocorticoid secretion.
Interestingly, by studying the relationship between expressed ADM and serum aldosterone concentration, we found a strong negative correlation in WT mice (Fig. 2E). Thus, linear regression found a -5.3 ± 1.0 slope (mean ± S.D.) with r2 = 0.84. Correlation Spearman test found a Ispearman = - 0.86 (P=0.024). On the contrary, this relationship was lacking in the Admhi/hi mice, suggesting that the strong relationship between aldosterone secretion and ADM production observed in WT animals was abolished in Admhi/hi mice. To better understand the regulatory impact of ADM on steroidogenesis, we conducted in vitro experiments on two human adrenal cell models.
Cortisol
Aldosterone
fold induction above control
10-
fold induction above control
100-
**
8
75
**
6-
*
50
4-
25
2
0
0-
Control
FK
K10
Angli
Control
FK
K10
Angll
CYP11B1
CYP11B2
fold induction above control
15-
fold induction above control
12
*
10-
*
8-
**
*
5-
4-
0
-
0
Control
FK
K10
Angli
Control
FK
K10
Angll
Comparison of adrenocortical carcinoma cells responses to various stimuli
Since H295R cellular responses to different agonists have been described as highly variable according to culture conditions (Rainey et al. 2004), we first examined the experimental conditions in which this cell model was responding to classical stimulations. Figure 3 presents how the H295R cells are able to respond to FK (20 uM), potassium (10 mM), and Ang II (1 µM). As anticipated, steroid overproductions are observed on the secretions of aldosterone (mean-fold induction over control ± S.D .: 26.5 ± 8.2 with FK; 45.2 ± 25.5 with K; 22.6 ± 17.4 with Ang II) and of cortisol (6.0 ± 1.9 with FK; 4.1 ± 1.9 with K; 2.7 ± 0.7 with Ang II) after 24 h stimulation while CYP11B2 (3.4 ± 1.8 with FK; 5.9 ± 3.1 with K; 4.9 ± 1.7 with Ang II) and CYP11B1 expressions (9.7 ± 2.4 with FK; 3.5 ± 1.5
Cortisol
Aldosterone
800
*
1.50
**
1.25
600
ng/ml
ng/ml
1.00
400
0.75
0.50
200-
0.25
0-
E
0.00
H295R
HAC15
H295R
HAC15
CYP11B1
CYP11B2
0.010
NS
0.06
**
Transcript expression
Transcript expression
0.04
0.005
0.02
0.000
0.00
H295R
HAC15
H295R
HAC15
with K; 5.0± 1.2 with Ang II) were significantly increased in H295R cells after 3 h incubation.
Although H295R cells appeared to be a good model to investigate the regulation of corticosteroid synthesis, the mineralocorticoid pathway remains poorly expressed, as very little basal aldosterone is secreted and scarce quantity of CYP11B2 transcript is found.
We tested another human adrenal cell model, derived from H295R cells: the HAC15 cells, known to produce more mineralocorticoids (Parmar et al. 2008). Indeed, we found, under basal conditions, a higher secretion of cortisol (over 14-times higher, P=0.028) and aldosterone (over 31-times higher, P=0.004), along with a higher expression of CYP11B2 (P=0.008) compared to H295R cells (Fig. 4).
CLR
H295R
HAC15
SMC
MCF7
Water
+
+
+
+
250 bp
150 bp
50 bp
RAMP2
H295R
HAC15
SMC
MCF7
Water
+
+
+
+
250 bp
150 bp
50 bp
B-actin
H295R
HAC15
SMC
MCF7
Water
+
+
+
+
250 bp
150 bp
-
50 bp
However, H295R and HAC15 cells showed no significant difference in CYP11B1 expression.
Adrenocortical carcinoma cells are sensitive to ADM
Furthermore, we tested whether H295R and HAC15 cells were able to express ADM complex receptor. Identification of ADM complex receptor (CLR+RAMP2) was performed by classical RT-PCR and agarose gel electrophoresis,
and breast cancer MCF7 cells were used as a negative control. Both cell lines express the ADM-receptor complex and, therefore, should be also able to respond to ADM treatment (Fig. 5). Therefore, we decided to pursue our study on both H295R and HAC15 cells.
ADM inhibits steroidogenesis in vitro
H295R and HAC15 cells were cultured on collagen I-coated dishes and stimulated with ADM (1 µM) or FK (20 µM) alone or in combination. Cortisol and aldosterone secretions were quantified by LC-MS/MS in the supernatants after 24 h stimulation and CYP11B1 and CPY11B2 expression quantified by RT-qPCR after 3 h stimulation.
ADM alone failed to show any effect on steroid secretions nor on CYP11B1 and CYP11B2 expression on both cell lines (Fig. 6).
However, regarding glucocorticoid production, ADM reduced FK-induced cortisol secretion in both H295R cells (6.4-fold vs 4.2-fold, P=0.02; Fig. 6A) and HAC15 cells (4.9-fold vs 2.5-fold, P=0.04; Fig. 6D). The cortisol-to-11- deoxycortisol (F/S) ratio, assessing the P450c11ß activity, significantly increased with FK in H295R cells and HAC15 cells (6.5-fold, P < 0.0001; Fig. 6B and 5.2-fold, P < 0.0001; Fig. 6E), respectively. Moreover, ADM significantly reduced this FK-induced stimulation of F/S ratio in H295R cells (3.4- fold, P=0.04; Fig. 6B) and in HAC15 cells (3.0-fold, P=0.03; Fig. 6E). Similarly, ADM reduced FK-induced CYP11B1 expression in both H295R (19.8-fold vs 8.6, P=0.04; Fig. 6C) and HAC15 cells (6.5-fold vs. 2.7-fold, P=0.03; Fig. 6F).
However, both H295R and HAC15 cells did not allow us to elucidate the effects of ADM on the mineralocorticoid synthesis pathway as supported by the literature, whether in a positive or negative manner. Thus, no effect of ADM was observed neither on aldosterone secretion nor on CYP11B2 expression (data not shown).
Discussion
Prematurity, accounting for 10% of births worldwide, is linked to numerous risks and complications as a result of organ immaturity (Frey & Klebanoff 2016). Steroidogenesis is one of the many biochemical processes impacted by prematurity, with a partial defect in steroid secretion at birth (Travers et al. 2017a) along with a partial yet transient defect in 11-hydroxylase activity despite the fact that P450c118 is expressed in the fetal adrenal cortex (Naccache et al. 2016) but never evaluated according to
H295R cells
A
Cortisol
B
EAlp450c118 (F/S)
C
CYP11B1
fold induction above control
10-
*
fold induction above control
15-
fold induction above control
40-
*
*
8-
30-
6-
10-
20-
4-
₼
10-
中
5-
2-
₲
-
0-
-
0
-
-
Control
ADM
0
FK
FK + ADM
Control
ADM
FK
FK + ADM
Control
ADM
FK
FK + ADM
HAC15 cells
D
Cortisol
E
EAI
(F/S)
F
CYP11B1
fold induction above control
P450c11B (
8
*
fold induction above control
8-
fold induction above control
12-
*
*
10-
6-
6-
₮
8
4.
4-
6
I
2-
4-
2-
2-
0
0
Control
ADM
FK
FK + ADM
Control
ADM
FK
0
FK+ADM
Control
ADM
FK
FK+ADM
the prematurity degree. Many regulators of corticosteroid synthesis have been thoroughly described (hypothalamic- pituitary axis (HPA), RAAS, kalemia), while some others have been recently discovered: bile acids (Liu et al. 2019), catecholamines (Mokuda et al. 1992), serotonin (Lefebvre et al. 2001), substance P (Wils et al. 2020), or adrenomedullin (ADM) (Kita et al. 2010).
To our knowledge, the effects of ADM on glucocorticoid production have not been studied, and those upon mineralocorticoid synthesis remain largely controversial.
The present study, performed on the cord-blood sample leftovers of the PREMALDO cohort, reveals that MR-proADM secretion is negatively correlated with gestational age. This biomarker has been chosen for its stability with higher plasma half-life than ADM itself, and for its reflection of ADM secretion (Schönauer et al. 2017). Previous papers already reported an increase of ADM in preterm neonates with intracerebral hemorrhage (Gazzolo et al. 2001), with bronchopulmonary dysplasia (Gong et al. 2020), or with perinatal infection (Admaty et al. 2012). It is also well established that ADM levels are higher in neonates than in adults (Koch et al. 2011) and that they normalized 2 days after birth in term neonates but remain elevated in preterm infants (Admaty et al. 2012). The biological function of ADM during human gestation, mostly synthesized by the placenta, remains to be fully elucidated. However, it is clear that ADM is required for
placental vascular tonicity (Di Iorio et al. 1997), neonatal vascular adaptation (Boldt et al. 1998, Rudolph 1998), and local regulation of the immune system (Li et al. 2013).
Regarding cortisol synthesis, we highlight a strong negative correlation between ADM and cortisol productions, along with a similar negative relationship between ADM levels and F/S ratio, which constitutes an index of 11-hydroxylase activity. Investigation of a mouse model overexpressing ADM shows a decrease of the B/DOC ratio only in Admhi/hi mice compared to WT littermates. These results are strongly in favor of a diminished 11-hydroxylase activity linked to ADM overexpression. Finally, in both human H295R and HAC15 cell models of corticosteroidogenesis, we demonstrate an inhibitory effect of ADM on cortisol secretion induced by FK associated with an inhibition of CYP11B1 expression.
Glucocorticoid synthesis may be impacted by ADM at several levels. Even though our LC-MS/MS method did not allow us to quantify pregnenolone nor 17-hydroxypregnenolone, we could not exclude that ADM could inhibit 3-hydroxysteroid dehydrogenase activity. The synthesis of ADM is clearly ubiquitous in humans, including in the adrenal glands, making even more complex the interpretation of ADM effects on the HPA axis. In vitro, ADM inhibits ACTH secretion (Samson et al. 1995), an effect confirmed in vivo in sheep (Parkes & May 1995). In late gestation, the placenta secretes corticotrophin-releasing
Effects of ADM on glucocorticoid production according to studied model
5 Human adrenocortical cells (H295R and HAC15)
Newborns
Overexpressing mice
☐ Very preterms show higher levels of MR- proADM at birth
☐ ADM overexpressing mice show lower 11ß- hydroxylase activity
☐ ADM alone has no effect on cortisol production
☐ O MR-proADM secretion is negatively linked to:
☐ When stimulated by FK, ADM inhibits:
☐ Cortisol secretion
☐ Gestational age
☐ 11ß-hydroxylase activity
☐ Cortisol production
☐ CYP11B1 transcription
☐ 11ß-hydroxylase activity
hormone (CRH) (Smith & Nicholson 2007). It remains plausible that the placental ADM regulates the placental CRH secretion, thereby affecting steroid synthesis, both in the placenta and the fetus. Labor and delivery constitute a highly stressful period, with elevated circulating cortisol levels (Miller et al. 2019). It is likely that ADM plays a role in the regulation of the stress responses related to this critical period, as it has also been shown that ADM levels increase during labor (Boldt et al. 1998). It is worth considering that prenatal betamethasone administration is often given to pregnant mothers at risk of preterm delivery to facilitate lung maturation of the fetuses and ultimately to reduce the mortality of the very preterm neonates. However, this glucocorticoid administration could contribute to the HPA and corticosteroid synthesis suppression observed at birth in preterm neonates (Ng et al. 1997, Karlsson et al. 2000, Roberts et al. 2017) which cannot, therefore, be explained only by elevated ADM concentrations.
ADM alone showed no effect, whereas when used in combination with a steroidogenesis-stimulating compound, it clearly inhibits CYP11B1 expression, reducing P450c116 activity and decreasing cortisol secretion. Figure 7 summarizes our main findings of ADM effects on glucocorticoid synthesis among the three models studied here (human newborns, ADM-overexpressing mice, and two human adrenocortical cell lines).
In 2003, Charles et al. found that ADM decreased the Ang II-induced aldosterone secretion. Similarly, an aldosterone decrease along with an increased plasma renin activity was found (Charles et al. 2003), consistent
with the increase of aldosterone-to-renin ratio found in preterm neonates of PREMALDO cohort (Martinerie et al. 2015). However, we failed to observe an inhibitory effect of ADM regardless of the model used. Similarly, in 2000, Lainchbury et al. also reported an increase of renin secretion in healthy volunteers under ADM perfusion, without any modification of aldosterone levels (Lainchbury et al. 2000). Nevertheless, we observed a strong negative correlation between ADM and plasma aldosterone in the WT control mice. Such a negative relationship between ADM and ARR has been also highlighted in healthy volunteers of the KORA F4 study (Then et al. 2016a,b). ADM perfusion was shown to decrease aldosterone secretion in patients with primary aldosteronism as well as in control subjects (Kita et al. 2010).
Otherwise, in our mouse model, genetically engineered mice overexpressing ADM present with a paradoxical increase of aldosterone secretion. It has also been reported that ADM could increase steroid secretion (Thomson et al. 2001) and aldosterone synthase expression in H295R cells (Thomson et al. 2001). On the contrary, inhibition of mineralocorticoid production by ADM has also been reported, in the rat both ex vivo and in vivo (32- 34). Collectively, our results did not provide any conclusive effect of ADM on mineralocorticoid synthesis, yet this topic remains to be further examined in other studies.
In ourmouse model, theloss of the relationshipbetween ADM levels and serum aldosterone concentrations in Admhi/hi mice is quite intriguing. It appears that aldosterone synthesis regulation could be different according to an
acute or chronic ADM production and its duration, as previously reported for ACTH stimulation (Cozza et al. 1989). Thus, under a physiological state, ADM could exert a fine regulation of aldosterone secretion along with what we found for cortisol production, indicating an inhibitory effect of ADM. On the contrary, in a situation where ADM is overexpressed starting from early developmental stages, it is likely that an adaptive mechanism to the chronic ADM-induced vasodilatation may exist. Therefore, the continuous stimulation of RAAS leads to an increase in aldosterone production.
In conclusion, we provide the first evidence for a direct relationship between plasma ADM and corticosteroid levels in newborns according to their gestational age. We also report the first plasma steroid metabolome of a genetically engineered mouse model overexpressing ADM. Our findings support an inhibitory effect of ADM at least on glucocorticoid synthesis. The strong negative correlation found in newborns and investigations on human adrenocortical cells further validate our hypothesis of a functional link between elevated ADM concentrations in preterm neonates and impaired 11-hydroxylase activity.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector.
Author contribution statement
M L and E P: these senior authors contributed equally to this work.
Acknowledgements
The authors thank HAC Pharma (P1156.0) for its financial support notably QYX during this work, and Dr Jérôme Bouligand for his help on microarray data interpretation during reviewing process.
References
Admaty D, Benzing J, Burkhardt T, Lapaire O, Hegi L, Szinnai G, Morgenthaler NG, Bucher HU, Bührer C & Wellmann S 2012 Plasma midregional proadrenomedullin in newborn infants: impact of prematurity and perinatal infection. Pediatric Research 72 70-76. (https://doi.org/10.1038/pr.2012.38)
Boldt T, Luukkainen P, Fyhrquist F, Pohjavuori M & Andersson S 1998 Birth stress increases adrenomedullin in the newborn. Acta Paediatrica 87 93-94. (https://doi.org/10.1080/08035259850157949)
Caron KM & Smithies O 2001 Extreme hydrops fetalis and cardiovascular abnormalities in mice lacking a functional adrenomedullin gene. PNAS 98 615-619. (https://doi.org/10.1073/pnas.021548898)
Caruhel P, Mazier C, Kunde J, Morgenthaler NG & Darbouret B 2009 Homogeneous time-resolved fluoroimmunoassay for the measurement of midregional proadrenomedullin in plasma on the fully automated system brahms kryptor. Clinical Biochemistry 42 725-728. (https://doi. org/10.1016/j.clinbiochem.2009.01.002)
Charles CJ, Lainchbury JG, Nicholls MG, Rademaker MT, Richards AM & Troughton RW 2003 Adrenomedullin and the renin-angiotensin- aldosterone system. Regulatory Peptides 112 41-49. (https://doi. org/10.1016/s0167-0115(03)00021-1)
Cozza EN, Ceballos NR & Lantos CP 1989 Effects of ACTH on the last step of aldosterone biosynthesis. Journal of Steroid Biochemistry 33 1253-1255. (https://doi.org/10.1016/0022-4731(89)90438-x)
De Martin S, Paliuri G, Belloni A, Orso G, Zanarella E, Stellin G, Milanesi O, Basso G, Ruga EM, Frasson C, et al. 2014 Expression and distribution of the adrenomedullin system in newborn human thymus. PLoS ONE 9 e97592. (https://doi.org/10.1371/journal.pone.0097592)
Di Iorio RD, Marinoni E, Scavo D, Letizia C & Cosmi EV 1997 Adrenomedullin in pregnancy. Lancet 349 328. (https://doi. org/10.1016/S0140-6736(05)62827-9)
Frey HA & Klebanoff MA 2016 The epidemiology, etiology, and costs of preterm birth. Seminars in Fetal and Neonatal Medicine 21 68-73. (https://doi.org/10.1016/j.siny.2015.12.011)
Gazzolo D, Marinoni E, Giovannini L, Letizia C, Serra G & Di Iorio R 2001 Circulating adrenomedullin is increased in preterm newborns developing intraventricular hemorrhage. Pediatric Research 50 544-547. (https://doi.org/10.1203/00006450-200110000-00020)
Gille J, Ostermann H, Dragu A & Sablotzki A 2017 MR-proADM: a new biomarker for early diagnosis of sepsis in burned patients. Journal of Burn Care and Research 38 290-298. (https://doi.org/10.1097/ BCR.0000000000000508)
Gong X, Qiu J, Qiu G & Cai C 2020 Adrenomedullin regulated by miRNA-574-3p protects premature infants with bronchopulmonary dysplasia. Bioscience Reports 40 BSR20191879. (https://doi.org/10.1042/ BSR20191879)
Holtbäck U & Aperia AC 2003 Molecular determinants of sodium and water balance during early human development. Seminars in Neonatology 8 291-299. (https://doi.org/10.1016/S1084-2756(03)00042-3)
Ishimitsu T, Kojima M, Kangawa K, Hino J, Matsuoka H, Kitamura K, Eto T & Matsuo H 1994 Genomic structure of human adrenomedullin gene. Biochemical and Biophysical Research Communications 203 631-639. (https://doi.org/10.1006/bbrc.1994.2229)
Jensen BL, Krämer BK & Kurtz A 1997 Adrenomedullin stimulates renin release and renin mRNA in mouse juxtaglomerular granular cells. Hypertension 29 1148-1155. (https://doi.org/10.1161/01.hyp.29.5.1148)
Karlsson R, Kallio J, Toppari J, Scheinin M & Kero P 2000 Antenatal and early postnatal dexamethasone treatment decreases cortisol secretion in preterm infants. Hormone Research 53 170-176. (https://doi. org/10.1159/000023563)
Kita T, Tokashiki M & Kitamura K 2010 Aldosterone antisecretagogue and antihypertensive actions of adrenomedullin in patients with primary aldosteronism. Hypertension Research 33 374-379. (https://doi. org/10.1038/hr.2010.8)
Kitamura K, Kato J, Kawamoto M, Tanaka M, Chino N, Kangawa K & Eto T 1998 The intermediate form of glycine-extended adrenomedullin is the major circulating molecular form in human plasma. Biochemical and Biophysical Research Communications 244 551-555. (https://doi. org/10.1006/bbrc.1998.8310)
Kitamura K, Kangawa K & Eto T 2002 Adrenomedullin and pamp: discovery, structures, and cardiovascular functions. Microscopy Research and Technique 57 3-13. (https://doi.org/10.1002/jemt.10052)
Journal of Endocrinology
Koch L, Dabek MT, Frommhold D & Poeschl J 2011 Stable precursor fragments of vasoactive peptides in umbilical cord blood of term and preterm infants. Hormone Research in Paediatrics 76 234-239. (https:// doi.org/10.1159/000329285)
Krintus M, Kozinski M, Braga F, Kubica J, Sypniewska G & Panteghini M 2018 Plasma midregional proadrenomedullin (MR-proADM) concentrations and their biological determinants in a reference population. Clinical Chemistry and Laboratory Medicine 56 1161-1168. (https://doi.org/10.1515/cclm-2017-1044)
Lainchbury JG, Troughton RW, Lewis LK, Yandle TG, Richards AM & Nicholls MG 2000 Hemodynamic, hormonal, and renal effects of short-term adrenomedullin infusion in healthy volunteers. Journal of Clinical Endocrinology and Metabolism 85 1016-1020. (https://doi. org/10.1210/jcem.85.3.6422)
Lefebvre H, Compagnon P, Contesse V, Delarue C, Thuillez C, Vaudry H & Kuhn JM 2001 Production and metabolism of serotonin (5-HT) by the human adrenal cortex: paracrine stimulation of aldosterone secretion by 5-HT. Journal of Clinical Endocrinology and Metabolism 86 5001-5007. (https://doi.org/10.1210/jcem.86.10.7917)
Li M, Schwerbrock NMJ, Lenhart PM, Fritz-Six KL, Kadmiel M, Christine KS, Kraus DM, Espenschied ST, Willcockson HH, Mack CP, et al. 2013 Fetal-derived adrenomedullin mediates the innate immune milieu of the placenta. Journal of Clinical Investigation 123 2408-2420. (https://doi.org/10.1172/JCI67039)
Liu L, Panzitt K, Racedo S, Wagner M, Platzer W, Zaufel A, Theiler- Schwetz V, Obermayer-Pietsch B, Müller H, Höfler G, et al. 2019 Bile acids increase steroidogenesis in cholemic mice and induce cortisol secretion in adrenocortical H295R cells via S1PR2, ERK and SF-1. Liver International 39 2112-2123. (https://doi.org/10.1111/liv.14052)
Marinoni E, Di Iorio R, Alò P, Villaccio B, Alberini A & Cosmi EV 1999 Immunohistochemical localization of adrenomedullin in fetal and neonatal lung. Pediatric Research 45 282-285. (https://doi. org/10.1203/00006450-199902000-00021)
Martinerie L, Pussard E, Yousef N, Cosson C, Lema I, Husseini K, Mur S, Lombès M & Boileau P 2015 Aldosterone-signaling defect exacerbates sodium wasting in very preterm neonates: the Premaldo study. Journal of Clinical Endocrinology and Metabolism 100 4074-4081. (https://doi. org/10.1210/jc.2015-2272)
Mazzocchi G, Musajo F, Neri G, Gottardo G & Nussdorfer GG 1996a Adrenomedullin stimulates steroid secretion by the isolated perfused rat adrenal gland in situ: comparison with calcitonin gene-related peptide effects. Peptides 17 853-857. (https://doi.org/10.1016/0196- 9781(96)00109-x)
Mazzocchi G, Rebuffat P, Gottardo G & Nussdorfer GG 1996b Adrenomedullin and calcitonin gene-related peptide inhibit aldosterone secretion in rats, acting via a common receptor. Life Sciences 58 839-844. (https://doi.org/10.1016/0024-3205(96)00017-3)
McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N, Solari R, Lee MG & Foord SM 1998 RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 393 333-339. (https://doi.org/10.1038/30666)
Miller WL & Auchus RJ 2011 The molecular biology, biochemistry, and physiology of human steroidogenesis and its disorders. Endocrine Reviews 32 81-151. (https://doi.org/10.1210/er.2010-0013)
Miller N, Asali AA, Agassi-Zaitler M, Neumark E, Eisenberg MM, Hadi E, Elbaz M, Pasternak Y, Fishman A & Biron-Shental T 2019 Physiological and psychological stress responses to labor and delivery as expressed by salivary cortisol: a prospective study. American Journal of Obstetrics and Gynecology 221 351.e1-351.e7. (https://doi.org/10.1016/j. ajog.2019.06.045)
Mokuda O, Sakamoto Y, Kawagoe R, Ubukata E & Shimizu N 1992 Epinephrine augments cortisol secretion from isolated perfused adrenal glands of guinea pigs. American Journal of Physiology 262 E806-E809. (https://doi.org/10.1152/ajpendo.1992.262.6.E806)
Naccache A, Louiset E, Duparc C, Laquerrière A, Patrier S, Renouf S, Gomez-Sanchez CE, Mukai K, Lefebvre H & Castanet M 2016 Temporal
and spatial distribution of mast cells and steroidogenic enzymes in the human fetal adrenal. Molecular and Cellular Endocrinology 434 69-80. (https://doi.org/10.1016/j.mce.2016.06.015)
Ng PC, Wong GW, Lam CW, Lee CH, Fok TF, Wong MY, Wong W & Chan DC 1997 Pituitary-adrenal suppression and recovery in preterm very low birth weight infants after dexamethasone treatment for bronchopulmonary dysplasia. Journal of Clinical Endocrinology and Metabolism 82 2429-2432. (https://doi.org/10.1210/jcem.82.8.4152)
Parkes DG & May CN 1995 ACTH-suppressive and vasodilator actions of adrenomedullin in conscious sheep. Journal of Neuroendocrinology 7 923-929. (https://doi.org/10.1111/j.1365-2826.1995.tb00737.x)
Parmar J, Key RE & Rainey WE 2008 Development of an adrenocorticotropin-responsive human adrenocortical carcinoma cell line. Journal of Clinical Endocrinology and Metabolism 93 4542-4546. (https://doi.org/10.1210/jc.2008-0903)
Pfaffl MW, Tichopad A, Prgomet C & Neuvians TP 2004 Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: best keeper excel-based tool using pair-wise correlations. Biotechnology Letters 26 509-515. (https://doi.org/10.1023/ b:bile.0000019559.84305.47)
Rainey WE, Saner K & Schimmer BP 2004 Adrenocortical cell lines. Molecular and Cellular Endocrinology 228 23-38. (https://doi. org/10.1016/j.mce.2003.12.020)
Risso FM, Sannia A, Gavilanes DAW, Vles HJ, Colivicchi M, Ricotti A, Li Volti G & Gazzolo D 2012 Biomarkers of brain damage in preterm infants. Journal of Maternal-Fetal and Neonatal Medicine 25 (Supplement 4) 101-104. (https://doi.org/10.3109/14767058.2012.715024)
Roberts D, Brown J, Medley N & Dalziel SR 2017 Antenatal corticosteroids for accelerating fetal lung maturation for women at risk of preterm birth. Cochrane Database of Systematic Reviews 3 CD004454. (https:// doi.org/10.1002/14651858.CD004454.pub3)
Rudolph AM 1998 Adrenomedullin: its role in perinatal adaptation. Acta Paediatrica 87 235-236. (https://doi.org/10.1080/08035259850157246) Samson WK, Murphy T & Schell DA 1995 A novel vasoactive peptide, adrenomedullin, inhibits pituitary adrenocorticotropin release. Endocrinology 136 2349-2352. (https://doi.org/10.1210/ endo.136.5.7720684)
Schiffer L, Anderko S, Hannemann F, Eiden-Plach A & Bernhardt R 2015 The CYP11B subfamily. Journal of Steroid Biochemistry and Molecular Biology 151 38-51. (https://doi.org/10.1016/j.jsbmb.2014.10.011)
Schönauer R, Els-Heindl S & Beck-Sickinger AG 2017 Adrenomedullin: new perspectives of a potent peptide hormone. Journal of Peptide Science 23 472-485. (https://doi.org/10.1002/psc.2953)
Smith R & Nicholson RC 2007 Corticotrophin releasing hormone and the timing of birth. Frontiers in Bioscience: Journal and Virtual Library 12 912-918. (https://doi.org/10.2741/2113)
Sulyok E, Varga F, Györy E, Jobst K & Csaba IF 1979 Postnatal development of renal sodium handling in premature infants. Journal of Pediatrics 95 787-792. (https://doi.org/10.1016/s0022-3476(79)80737-4)
Then C, Rottenkolber M, Lechner A, Meisinger C, Heier M, Koenig W, Peters A, Rathmann W, Bidlingmaier M, Reincke M, et al. 2016a Altered relation of the renin-aldosterone system and vasoactive peptides in type 2 diabetes: the KORA F4 study. Atherosclerosis 252 88-96. (https:// doi.org/10.1016/j.atherosclerosis.2016.07.905)
Then C, Rottenkolber M, Lechner A, Meisinger C, Heier M, Koenig W, Peters A, Rathmann W, Bidlingmaier M, Reincke M, et al. 2016b Dataset of the associations of aldosterone to renin ratio with MR-proANP and MR-proADM. Data in Brief 8 1395 - 1399 . (https://doi.org/10.1016/j. dib.2016.08.008)
Thomson LM, Kapas S, Carroll M & Hinson JP 2001 Autocrine role of adrenomedullin in the human adrenal cortex. Journal of Endocrinology 170 259-265. (https://doi.org/10.1677/joe.0.1700259)
Thomson LM, Kapas S & Hinson JP 2003 Paracrine effects of PAMP and adrenomedullin on the human adrenal H295R cell line: pamp but not adrenomedullin stimulates DHEA secretion. Regulatory Peptides 112 3-7. (https://doi.org/10.1016/S0167-0115(03)00016-8)
Journal of Endocrinology
Travers S, Martinerie L, Boileau P, Lombès M & Pussard E 2017a Alterations of adrenal steroidomic profiles in preterm infants at birth. Archives of Disease in Childhood: Fetal and Neonatal Edition 103 F143-F151. (https:// doi.org/10.1136/archdischild-2016-312457)
Travers S, Martinerie L, Bouvattier C, Boileau P, Lombès M & Pussard E 2017b Multiplexed steroid profiling of gluco- and mineralocorticoids pathways using a liquid chromatography tandem mass spectrometry method. Journal of Steroid Biochemistry and Molecular Biology 165 202-211. (https://doi.org/10.1016/j.jsbmb.2016.06.005)
Trincot CE, Xu W, Zhang H, Kulikauskas MR, Caranasos TG, Jensen BC, Sabine A, Petrova TV & Caron KM 2019 Adrenomedullin induces cardiac lymphangiogenesis after myocardial infarction and regulates cardiac edema via connexin 43. Circulation Research 124 101-113. (https://doi.org/10.1161/CIRCRESAHA.118.313835)
Valenzuela-Sánchez F, Valenzuela-Méndez B, Rodríguez-Gutiérrez JF, Estella-García Á & González-García MÁ 2016 New role of biomarkers: mid-regional pro-adrenomedullin, the biomarker of organ failure. Annals of Translational Medicine 4 329. (https://doi.org/10.21037/ atm.2016.08.65)
Viengchareun S, Kamenicky P, Teixeira M, Butlen D, Meduri G, Blanchard- Gutton N, Kurschat C, Lanel A, Martinerie L, Sztal-Mazer S, et al. 2009 Osmotic stress regulates mineralocorticoid receptor expression in a
novel aldosterone-sensitive cortical collecting duct cell line. Molecular Endocrinology 23 1948-1962. (https://doi.org/10.1210/me.2009-0095) Vigué B, Leblanc PE, Moati F, Pussard E, Foufa H, Rodrigues A,
Figueiredo S, Harrois A, Mazoit JX, Rafi H, et al. 2016 Mid-regional pro- adrenomedullin (MR-proADM), a marker of positive fluid balance in critically ill patients: results of the ENVOL study. Critical Care 20 363. (https://doi.org/10.1186/s13054-016-1540-x)
Wetzel-Strong SE, Li M, Klein KR, Nishikimi T & Caron KM 2014 Epicardial-derived adrenomedullin drives cardiac hyperplasia during embryogenesis. Developmental Dynamics 243 243-256. (https://doi. org/10.1002/dvdy.24065)
Wils J, Duparc C, Cailleux AF, Lopez AG, Guiheneuf C, Boutelet I, Boyer HG, Dubessy C, Cherifi S, Cauliez B, et al. 2020 The neuropeptide substance P regulates aldosterone secretion in human adrenals. Nature Communications 11 2673. (https://doi.org/10.1038/s41467-020-16470-8)
Yamaguchi T, Baba K, Doi Y & Yano K 1995 Effect of adrenomedullin on aldosterone secretion by dispersed rat adrenal zona glomerulosa cells. Life Sciences 56 379-387. (https://doi.org/10.1016/0024-3205(94)00903-1)
Yamaguchi T, Baba K, Doi Y, Yano K, Kitamura K & Eto T 1996 Inhibition of aldosterone production by adrenomedullin: a hypotensive peptide, in the rat. Hypertension 28 308-314. (https://doi.org/10.1161/01. hyp.28.2.308)
Received in final form 26 July 2021 Accepted 9 August 2021 Accepted Manuscript published online 9 August 2021