differential proteomic analysis of adrenal gland during postnatal development

RESEARCH ARTICLE

Differential proteomic analysis of adrenal gland during

postnatal development

Alberto Pascual1,2, Antonio Romero-Ruiz1 and Jose Lopez-Barneo1,2

1 Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocıo/CSIC/Universidad de Sevilla,Sevilla, Spain

2 Centro de Investigacion Biomedica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain

Received: September 21, 2008

Revised: January 18, 2009

Accepted: February 12, 2009

The adrenal glands (AGs) are endocrine organs essential for life. They undergo a fetal to adult

developmental maturation process, occurring in rats during the first postnatal month. The

molecular modifications underlying these ontogenic changes are essentially unknown. Here

we report the results of a comparative proteomic analysis performed on neonatal (Postnatal

day 3) versus adult (Postnatal day 30) AGs, searching for proteins with a relative higher

abundance at each age. We have identified a subset of proteins with relevant expression in

each developmental period using 2-DE and DIGE analysis. The identified proteins belong to

several functional categories, including proliferation/differentiation, cell metabolism, and

steroid biosynthesis. To study if the changes in the proteome are correlated with changes at

the mRNA level, we have randomly selected several proteins with differential expression and

measured their relative mRNA levels using quantitative RT-PCR. Cell-cycle regulating

proteins (retinoblastoma binding protein 9 and prohibitin) with contrasting effects on

proliferation are expressed differentially in neonatal and adult AG. Progesterone metaboliz-

ing enzymes, up-regulated in the neonatal gland, might contribute to the hyporesponsiveness

of the adrenal cortex characteristic of this developmental period. We have also observed in the

adult gland a marked up-regulation of enzymes involved in NAD(P)H production, thus

providing the reducing power necessary for steroid hormone biosynthesis.

Keywords:

Adrenal gland / DIGE / Postnatal development / Quantitative proteomics

1 Introduction

Adrenal glands (AGs) are complex endocrine organs

essential for life. Each gland is a combination of two sepa-

rate tissues: the outer zone, or cortex, comprising 80–90% of

the organ, and the inner zone, or medulla. The adult adrenal

cortex (AC) is a component of the hypothalamic-pituitary-

adrenal axis organized in three distinct cell layers. The outer

zona glomerulosa synthesizes mineralocorticoids (mainly

aldosterone) that control sodium and potassium balance.

The central zona fasciculata produces glucocorticoids

(cortisol and corticosterone) that regulate carbohydrate and

protein metabolism. The inner zona reticularis is specia-

lized in the production of sex steroids, which contribute to

establishing and maintaining secondary sexual character-

istics [1]. The adrenal medulla (AM) derives from neural

crest progenitors of the sympathoadrenal lineage and is the

source of catecholamine (CAT) hormones. CATs release

from the AM contributes to physiological adaptations to

stressful situations [1].

From a functional viewpoint the AG undergoes major

postnatal developmental changes. The AC shifts from

Abbreviations: AC, adrenal cortex; AG, adrenal gland; AM, adrenal

medulla; CAT, catecholamine; HSD, hydroxy steroid dehydrogen-

ase; NADPH, nicotinamide adenine dinucleotide phosphate; P3,

postnatal day 3; P30, postnatal day 30; Pel15, pellet from a 15000�g

centrifugation; Pel100, pellet from a 100000�g centrifugation; PGD,

phosphogluconate dehydrogenase; Rb, retinoblastoma; Sup100,

supernatant from a 100000�g centrifugation; VDAC, voltage-

dependent anion channel

Correspondence: Dr. Alberto Pascual, Instituto de Biomedicina

de Sevilla, Hospital Universitario Virgen del Rocıo/CSIC/Univer-

sidad de Sevilla, Sevilla 41013, Spain

E-mail: [email protected],

[email protected]

Fax: 134-954617301

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

2946 Proteomics 2009, 9, 2946–2954DOI 10.1002/pmic.200800748

a relatively hyporesponsive status during the neonatal

period to a fully functional secretory tissue during the first

30 days of animal life [2–4]. The unresponsive status

of corticosterone-secreting cells in early neonatal stages

favors maintenance of the low glucocorticoid levels

during the first postnatal days required for normal growth

[5], development of immune function (reviewed in [6]),

and the establishment of the correct adult neuronal cell

number [7]. In contrast, cells in the neonatal AM are

known to secrete CATs in response to numerous

stimuli (hypoxia, acidosis, hypercapnia, etc.). In the adult

AM, CATs are secreted to the circulation after neurogenic

stimulation through the splanchnic nerve [8]. It is

generally agreed that neonatal AM responsiveness to

these stimuli is necessary for fetal homeostasis and is also

critical for the metabolic, respiratory, and cardiovascular

changes necessary for the rapid adaptation to extrauterine

life [9–15].

Although the data summarized above indicate that AG is

an excellent example of an organ undergoing major

homeostatic postnatal functional modifications, the under-

lying molecular mechanisms are essentially unknown.

In order to identify some of the subjacent molecular

mechanisms of AG postnatal development, we have

performed a comparative proteomic analysis of neonatal

(Postnatal day 3, P3) versus adult (Postnatal day 30, P30) AGs

and here we report the initial characterization of some of the

molecular modifications identified. Protein profiles from

AGs fractions containing soluble proteins, organelles, and

large protein complexes are described; changes between the

perinatal and adult AGs are identified; and the subjacent

functions of the differentially expressed proteins are

discussed.

2 Materials and methods

2.1 Animals

Wistar Han rats were kept under 12:12 h cycle of light with

ad libitum access to food and drink. Neonatal pups (P3) were

killed by decapitation and AGs were quickly dissected and

frozen under liquid nitrogen. Adult rats (P30) were killed by

chloral hydrate injection and AGs were dissected and frozen

under liquid nitrogen. Up to 24 AGs were used in each P3

extract and 4 AGs from one female and one male rat were

pooled per P30 extract.

2.2 Cell fractioning

Pooled AGs were disrupted at 41C with cold hypotonic

buffer (10 mM MOPS, 85 mM sucrose, pH 7.2) in a Dounce

homogenizer, mixed with an equal volume of hypertonic

buffer (30 mM MOPS, 250 mM sucrose, pH 7.2) and

centrifuged at 600� g for 15 min at 41C. The pellet was

discarded and the supernatant was centrifuged again at

15 000� g for 15 min at 41C [16]. The pellet was resus-

pended in cold lysis buffer (30 mM Tris-HCl, 2 M thiourea,

7 M urea, 4% w/v CHAPS, pH 8.5) to constitute the pellet

from a 15 000� g centrifugation (Pel15) extract. The super-

natant was centrifuged at 100 000� g for 1 h at 41C and the

pellet was dissolved in lysis buffer (Pel100 extract). The

supernatant was mixed with an equal volume of lysis buffer

(Sup100 extract).

2.3 Western blot

Fifty micrograms of protein were loaded in 10% SDS poly-

acrylamide gels and immobilized into PVDF membranes.

Mouse anti-alpha-tubulin (Sigma, 1:10 000 dilution), rabbit

anti-Pan-Cadherin (AbCam, 1:200 dilution), and mouse

anti-voltage-dependent anion channel (VDAC) (Calbiochem,

1:3000 dilution) were used. Secondary antibodies conjugated

to HRP and ECL-plus (GE Healthcare) were used for signal

development. Images were acquired with Typhoon 9400 (GE

Healthcare).

2.4 Sample preparation and labelling

Pel15, Pel100, and Sup100 protein extracts were prepared

for 2-DE by using 2-D clean-up kit (GE Healthcare) follow-

ing manufacturer instructions. Precipitated proteins were

finally resuspended in lysis buffer. Protein was quantified

using RC-DC protein assay kit (Bio-Rad). pH was adjust to

8.0 by adding a few drops of 100 mM NaOH and 50 mg of

protein were labelled with 400 pmol of either Cy3 or Cy5

dyes (GE Healthcare). A 50 mg protein mix, containing equal

amount of each protein extract used in the experiment, was

labelled with 400 pmol of Cy2 dye (GE Healthcare). This mix

constituted the internal standard sample. Labelled samples

were immediately subjected to IEF.

2.5 2-DE analysis

A total 150 mg of labelled protein, containing three equal

amount extracts (P3, P30, and the internal standard), were

complemented with 0.5% IPG buffer, pH 3–11, 0.3% DTT,

and traces of bromophenol blue and applied on Immobili-

neTM DryStrip, pH 3–11, 18 cm strips (rehydrated in

Destreak Rehydratation Solution, GE Healthcare) using the

Ettan IPG-phor Cup loading Manifold (GE Healthcare).

Focusing was performed in the IPGphor IEF System in four

steps: 300 V–3 h step and hold, 3000 V–6 h gradient,

8000 V–3 h gradient, and 8000 V until steady state was

reached. IPG strips were incubated for 15 min in 50 mM

Tris-HCl, 6 M urea, 30% glycerol, 2% SDS, 10 mg/mL DTT,

pH 8.8, and another 15 min with 50 mM Tris-HCl, 6 M urea,

30% glycerol, 2% SDS, 25 mg/mL iodoacetamide, pH 8.8.

Proteomics 2009, 9, 2946–2954 2947


The second dimension was performed on 5–20% SDS-

PAGE gradient gels using SE-600 electrophoresis system

(GE Healthcare). Strips were cut by 1 cm at each end to

adjust to SE-600 width. For DIGE analysis, fluorescent gel

images were obtained using a Typhoon 9400 Scanner and

analyzed with the DeCyder Differential analysis software

(GE Healthcare) by applying default parameters.

For preparative IEF, Immobiline DryStrips were rehy-

drated in the presence of 0.5–1 mg of protein and 0.5% IPG

buffer, pH 3–11, 0.3% DTT, and Destreak reagent (GE

Healthcare). Focusing and second dimension were

performed as described before. Protein was detected with a

MALDI-MS(/MS) compatible silver (GE Healthcare) or

SYPRO (Bio-Rad) staining to allow subsequent protein

identification.

2.6 Total RNA extraction and quantitative RT-PCR

Total RNA from P3 and P30 AGs were extracted with TRIzol

reagent (Invitrogen) in a homogenizer (Omni 2000). RNA

samples (5 mg) were treated with DNase RNase free (GE

Healthcare) and copied to cDNA using SuperScriptII

reverse transcriptase (Invitrogen) in a final volume of 20 mL.

Real-time PCR was performed in an ABI Prism 7500

Sequence Detection System (Applied-Biosystems) using

SYBR Green PCR Master mix (Applied-Biosystems) and the

thermocycler conditions recommended by the manu-

facturer. PCRs were performed in duplicates in a total

volume of 25mL containing 1mL of the RT reaction. In each

sample 18S and b-act RNA levels were estimated to

normalize for RNA input amounts and to perform relative

quantifications. Primers were designed using the Primer

Express software (Applied-Biosystems) and are listed in

Supporting Information Table 3.

2.7 Statistical analysis

Data are presented as mean7SEM. and were analyzed with

Student’s t-test. po0.05 was considered statistically signifi-

cant.

Additional methodological description is presented as

Supporting Information.

3 Results

3.1 Characterization of AG cellular fractions

To decrease sample complexity before 2-DE protein

separation, a centrifugation-based cell fractioning protocol

was performed (see Section 2). The protocol has been

described for mitochondrial separation [16] and a few

modifications were made. To obtain a fully soluble fraction

from the Sup15, an ultracentrifugation step was performed.

Pel15 is expected to contain organellar proteins, Sup100 is

expected to contain mainly soluble proteins and Pel100 has

been described as a fraction enriched in macro-protein

complexes such as ribosome and proteosome. To estimate

the protein composition of each fraction, we selected three

subcellular markers. We performed Western blot detection

of alpha-tubulin as a soluble marker, VDAC as a mito-

chondrial probe and pan-cadherin as a plasma membrane

indicator to validate if our experimental approach generated

similar fractions from neonatal and adult AG extracts. The

three proteins were similarly distributed in fractions from

the neonatal and adult AGs (Fig. 1). At both developmental

stages, Sup100 was enriched in soluble proteins, as revealed

by the presence of relatively high levels of alpha-tubulin

(Fig. 1); Pel15 was enriched in organellar and membrane

proteins, as shown by VDAC and Pan-Cad detection; and

Pel100 resulted in a fraction where the three markers were

detected. The plasma membrane marker was present in all

fractions (Fig. 1). Therefore, our protocol produced fractions

with equivalent qualitative protein composition in the two

developmental stages analyzed, allowing for proper

comparative analysis.

3.2 2-DE differential analyses of adrenal postnatal

ontogeny

To identify proteins with a different expression pattern

during postnatal AG development, we performed quantita-

tive 2-DE using Ettan DIGE technology (GE Healthcare).

Four replicates of neonatal and adult extracts were fractio-

nated, labeled and separated as described in Section 2. A

representative gel for each fraction is shown in Fig. 2. We

have resolved in 2-D-gels 2587 spots from the Sup100

Sup100 Pel100Pel15

Neonatal (P3) Adult (P30)

Pan-cad

-Tub

VDAC

120 kDa

31 kDa

55 kDa

Sup100 Pel100Pel15

Figure 1. Fractionation of neonatal and adult AG

produces equivalent protein extracts. Western

blot analysis of different cellular fractions

(Sup100, Pel15, and Pel100). Extracts from

neonatal (P3, left) and adult (P30, right) AG

were analyzed by Western blot using anti-a-

tubulin, VDAC, and pan-cad antibodies. The two

postnatal stages (top) and the different fraction

extracts loaded are shown (bottom). Molecular

weights are showed on the right-hand side and

the antibodies used in each panel are shown on

the left-hand side.

2948 A. Pascual et al. Proteomics 2009, 9, 2946–2954


fraction (Fig. 2A); 2342 from the Pel15 extracts (Fig. 2B);

and 2281 from the Pel100 fraction (Fig. 2C). Spots with a

differential expression during postnatal development were

identified with DeCyder software. Three different extracts

were separated in each 2-D-gel. Neonatal (extract 1, labeled

with Cy3 or Cy5) and adult (extract 2, labeled with Cy3 or

Cy5) spots were normalized to an internal standard (extract

3, Cy2 labeled) containing equal amount of each protein

extract used in the experiment. After in-gel normalization,

the four replicates were analyzed to describe differences in

expression. The criteria for spot inclusion in the protein of

interest list were as follows: (i) a change of expression of at

least 1.9-fold, (ii) a t-test with po0.05, and (iii) the identifi-

cation of the spots in the four experimental replicates.

Proteins with differential expression are encircled in Fig. 2

and Supporting Information Fig. 2. We detected 178 spots

with an increased level in the neonatal AG and 200 spots

with a higher representation in the adult AG. Proteins of

interest successfully identified by MALDI-MS(/MS) are

indicated in Fig. 2 and listed in Supporting Information

Table 1. In Supporting Information Fig. 2 and Supporting

Information Table 2 are compiled proteins whose expres-

sion was changed without a positive identification. In total,

we have identified 131 spots representative of 101 proteins.

The overall coverage in our experiment is estimated to be

around 60–70% of the protein changes.

3.3 Ontogenic classification

Identified proteins were classified using ‘‘the gene ontology

(GO) project’’ terms corresponding to the molecular function

category (http://www.geneontology.org/). The analysis of the

abundance of each ontological categories revealed substantial

functional differences between neonatal and adult AGs. The

contribution at both developmental time points of each

functional category is schematically depicted in Fig. 3.

A7

2977 532

78 27

2 10111481 83 15 31

17 2620

13 22

23192186 88 2497

30 9575

1 90 8533

18

28

16

799 894 9684 12

91

3

92

116

258287

80 8993

4

76

Merge

Neonatal (P3)Adult (P30)

C 62 56725167

4971 6668 57

5365 54126

486069

123

70

63 55 59116

124

120

12152

61 127 11958128

5047

6474

73 125117129 130

122

131

118

115


B 34

100 11199 110

108 1051134441 112

43104

4540

3946

107101 37

38 98

3536 42

102114

109

103106


Merge

Merge

Sup100

Pel15

Pel100

Figure 2. 2-DE maps of rat AG fractions. Representative images

of 2-DE are shown for Sup100 (A), Pel15 (B), and Pel100 protein

extracts (C). Encircled spots were identified by MALDI-MS/MS.

The spot numbers correspond to numbering in Supporting

Information Table 1. For simplicity the internal standard extract

is not showed in the image (Cy2-labeled proteins). The P3

(neonatal) extract was labeled with Cy3 (red) in (A) and with Cy5

in (B) and (C) (green). P30 (adult) extracts were labeled with Cy5

in (A) and with Cy3 in (B) and (C).

Binding and TransportEnzyme inhibitor

TransferaseIsomeraseLigaseHydrolase

Signal transducerLyaseSmall protein activatingenzymeDeaminase

StructuralRNA processing

Oxidoreductase

A

B

Neonatal (P3)

Adult (P30)

Figure 3. Classification of the identified proteins by molecular

function. Proteins identified were classified by molecular func-

tion and represented graphically. The different categories were

assigned using the molecular function terms from the AmiGO

gene ontology web server. (A) Distribution by categories of

neonatal proteins. (B) Categorization of adult proteins.

Proteomics 2009, 9, 2946–2954 2949


3.4 Quantitative RT-PCR analysis

In order to confirm the changes described at the protein

level, we selected proteins differentially regulated at the two

developmental stages and quantified their cognate mRNA

expression by quantitative RT-PCR. We studied relative

mRNA levels of methylcrotonoyl-CoA carboxylase 1 (Mccc1);

propionyl CoA carboxilase beta (Pccb); Aldo-keto reductasefamily 1, member C18 (20ahydroxysteroid dehydrogenase

(HSD), Akr1c18); and ferritin light chain 1 (Ftl1). As shown in

Supporting Information Fig. 1A, the mRNA dynamics

during postnatal ontogeny reproduces the changes descri-

bed at the protein levels for Mccc1, Akr1c18, and Ftl1.

However, Pccb protein shows higher expression at P3

(Supporting Information Table 1) and higher mRNA levels

at P30 (Supporting Information Fig. 1A). 20aHSD expres-

sion has been reported during the development of female

and male mice AGs [17]. In that study, male expression was

restricted to the third week of age and, in contrast, observed

in all time points (P10–17 wk) in females. To check whether

these sex differences were also observed in rats, we analyzed

male and female 20aHSD expression levels at P3 and P30.

mRNA expression was higher in both sexes at P3 and a

strong decay was observed at P30 in both groups (Support-

ing Information Fig. 1B). Therefore, 20aHSD expression

level was similar at the early postnatal days in male and

female AGs.

4 Discussion

The rodent AG acquires its final organ morphology and

function during the first postnatal month. The adrenal cells

proliferate, migrate, and differentiate during this develop-

mental period to produce the characteristic layers of adult

AGs. By around P30, rat AG is terminally differentiated at

both functional and anatomical levels. AC cells (particularly

the corticosterone-secreting cells) are unresponsive in early

neonatal stages, which favors maintenance of the low

glucocorticoid levels during the first postnatal days required

for normal growth [5], development of immune function

(reviewed in [6]) and the establishment of the correct adult

neuronal cell number [7]. On the other hand, neonatal

chromaffin cells in the AM show non-neurogenic respon-

siveness to O2 and CO2 tension as well as other stressful

stimuli to facilitate the metabolic, respiratory, and cardio-

vascular changes required for the rapid adaptation of the

neonate to extrauterine life [9, 12]. This direct chemosensi-

tivity disappears in the adult AM [8, 13]. In order to elucidate

the molecular mechanisms associated with the AG devel-

opmental maturation we present here the data obtained

from a comparative proteomic analysis of neonatal versusadult AGs. This experimental approach has revealed chan-

ges in the amount of specific proteins in each develop-

mental period. Among the proteins involved in acute AG

responses to stress, we have identified proteins associated

with chromaffin granule release (chromogranin B and pro-

opiomelanocortin, Fig. 2 and Table 1), which were respec-

tively up-regulated 2.8-fold in adult and 1.9-fold in neonatal

AGs. Chromogranine up-regulation may simply reflect the

increase in size and transmitter content of the adult AG,

whereas the decrease in pro-opiomelanocortin in adult

adrenal cells could be due to the shift of this pro-hormone

production from AG (in neonate) to the hypophysis (in the

adult). On the other hand, we have not observed changes in

proteins that could be ascribed to perinatal O2 or CO2

sensitivity of AM cells. These changes probably depend on

ion channels and other transmembrane proteins, which

could not be studied here due to the limitations of 2-DE

analysis to resolve hydrophobic proteins.

Several biochemical pathways previously suspected to be

regulated by AG maturation are recapitulated in this study.

Among the proteins regulated, we have focused our atten-

tion on those involved in steroidogenesis, cell proliferation,

and cholesterol synthesis. In addition, we have also observed

that AG maturation is accompanied by coordinated meta-

bolic modifications that facilitate the adult functional role of

the gland, especially the AC.

Regarding the several routes of steroidogenesis, two

enzymes (20aHSD and 3aHSD) are highly expressed in

neonatal but presented low expression levels in adult AGs

(Fig. 2 and Table 1). The exact functional role of these

progesterone-metabolizing enzymes is not fully understood.

The primary, nonredundant function of 20aHSD estab-

lished from the study of 20aHSD deficiency is to suppress

progesterone levels in the ovary that is necessary for

parturition to take place [18]. 20aHSD has been recently

described as a marker of murine X-zone [17] and a previous

work has proved that both 20- and 3aHSD enzymatic

activities are down-regulated in cancer breast lines [19]. This

down regulation is thought to mediate the increase in

proliferation and the decrease in attachment associated with

tumor progression [20, 21]. The fact that 20aHSD and

3aHSD are dramatically up-regulated in the AG during the

first postnatal days, at both the protein (see Fig. 2 and

Table 1) and mRNA (20aHSD, see Supporting information

Fig. 1) levels, suggests a role of these enzymes in controlling

AG cell proliferation. The coordinated developmental regu-

lation of both proteins and their similar enzymatic activities

suggest that they have a common role during AG ontogeny.

This could explain the absence of phenotype in the 20aHSD

knockout mice [17]. Besides the control of cell proliferation,

an additional function for 3- and 20aHSD could be to reduce

the availability of progesterone during the first days after

birth, a function that has been shown for 20aHSD in the

ovary [18], thus contributing to the low responsiveness of

AGs to stress during this specific developmental period.

Besides the changes in progesterone metabolizing

enzymes, other proteins related with proliferation have

emerged from this study. The retinoblastoma (Rb) tumor

suppressor protein is a major regulator of the mammalian

cell cycle, suppressing cell growth by binding the E2F family



Table 1. Proteins identified and addressed in Section 4

IDa) Protein AC Protein name (gene name) t-Test valueb) Foldchange

GO termc)

Proteins up-regulated in adult versus neonatal AGs

48 NP_036686 Enolase 1, alpha (Eno1) 0.000061 3.46 Lyase50 NP_001017461 Similar to oxoglutarate dehydrogenase, lipoamide

(LOC360975)0.0012 3.31 Oxidoreductase

51 AAH64655 Enoyl coenzyme A hydratase, short chain, 1, mitochondrial(Echs1)

0.0000026 3.19 Lyase

34 NP_036658 Chromogranin B (Chgb) 0.0012 2.82 Binding andtransport

4 NP_445742 Phosphoglycerate mutase 1 (Pgam1) 0.000025 2.7 Isomerase5 NP_001009653 Methylcrotonoyl-coenzyme A carboxylase 1, alpha (Mccc1) 0.0000082 2.69 Ligase55 NP_955417 Dihydrolipoamide dehydrogenase (Dil) 0.0002 2.53 Oxidoreductase57 NP_001019409 Glutamyl-prolyl-tRNA synthetase (Eprs) 0.003 2.45 Binding and

transport59 NP_001007621 Pyruvate dehydrogenase, lipoamide, beta (Pdhb) 0.00037 2.45 Oxidoreductase8 NP_476534 L-3-hydroxyacyl–coenzyme A dehydrogenase, short chain

(Hadhsc)0.000012 2.42 Oxidoreductase

9 AAH88396 Glycerol-3-phosphate dehydrogenase 1, soluble (Gpd1) 0.000075 2.42 Oxidoreductase37 NP_058704 Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) 0.0025 2.38 Oxidoreductase60 AAH87651 Phosphoglycerate kinase 1 (Pgk1) 0.00021 2.33 Transferase14 NP_001012177 Methylcrotonoyl-coenzyme A carboxylase 2, beta (Mccc2) 0.000095 2.29 Ligase65 NP_446278 Pyruvate dehydrogenase kinase, isoenzyme 1 (Pdk1) 0.0014 2.28 Transferase39 NP_037309 Glutamate oxaloacetate transaminase 2, mitochondrial (Got2) 0.0072 2.22 Transferase19 NP_058701 Fumarate hydratase 1(Fh1) 0.000013 2.15 Lyase22 NP_001100396 Similar to A1325464 protein, predicted

(RGD1307976_predicted)0.000026 2.11 Oxidoreductase

23 NP_077067 Ferredoxin reductase (Fdxr) 0.00047 2.1 Oxidoreductase24 NP_036724 Isovaleryi coenzyme A dehydrogenase (Ivd) 0.00000086 2.06 Oxidoreductase74 NP_001009637 Leucyl-tRNA synthetase (Lars) 0.0065 2.02 Binding and

transport41 AAP92648 Phosphogluconate dehydrogenase (Pgd) 0.000066 1.99 Oxidoreductase29 XP_236965 Similar to Mut protein, predicted (RGD1564912_predicted) 0.00000056 1.96 Oxidoreductase30 NP_446090 Isocitrate dehydrogenase 3 (NAD1) alpha (idh3a) 0.0000068 1.94 Oxidoreductase31 XP_001073523 Similar to 3-oxoacid CoA transferase 1 (LOC690163) 0.0000002 1.94 Transferase32 AAA88512 Propionyl-coenzyme A carboxylase, alpha polypeptide

(Pcca)0.000019 1.94 Ligase

43 XP_001053031 Prohibitin (Phb) 0.007 1.93 Binding andtransport

Proteins up-regulated in neonatal versus adult AGs

77 NP_612519 Aldo-keto reductase family 1, member C18 (Akr1c18) 0.000027 17.03 Oxidoreductase81 NP_612556 3-Alpha-hydroxysteroid dehydrogenase (LOC191574) 0.0000087 4.4 Oxidoreductase82 NP_665885 Fatty acid binding protein 5, epidermal (Fabp5) 0.000000049 4.23 Binding and

transport83 NP_058726 Propionyl coenzyme A carboxylase, beta polypeptide

(Pccb)0.00022 3.17 Ligase

121 NP_001006982 Dihydrolipoamide S-succinyltransferase, E2 component(Dist)

0.0000083 3.04 Transferase

84 NP_062092 Retinoblastoma binding protein 9 (Rbbp9) 0.00012 2.81 Binding andtransport

95 NP_001433 Fatty acid binding protein 4, adipocyte (Fabp4) 0.0004 2.13 Binding andtransport

96 XP_001081395 Phenylethanolamine-N-methyltransferase (Pnml) 0.00066 2.05 Transferase113 NP_037290 Dopamine beta hydroxylase (Dbh) 0.0022 1.97 Hydroxylase98 XP_001066558 Similar to Glucosamine-6-phosphate isomease

(LOC683570)0.0011 1.9 Isomerase

116 NP_647542 Pro-opiomelanocortin (Pomc) 0.00035 1.9 Binding andtransport

a) ID numbers correspond to spots encircled in Fig. 2.b) The p values of paired t-test.c) Ontogenic classification according to the molecular function category.

Proteomics 2009, 9, 2946–2954 2951


of transcription factors. Agents that disrupt the interaction

between Rb family proteins and E2F promote cell prolifera-

tion. Rb-binding protein 9 is an oncogenic protein able to

compete with E2F for Rb binding [22]. In our study, we have

shown that Rb-binding protein 9 is up-regulated in neonatal

stages, suggesting a tendency to cell proliferation in this

developmental period. In contraposition the prohibitin, a

protein that reinforces the blocking of cell cycle progression

caused by Rb protein, is up-regulated in adult AG, suggesting

a slow down in proliferation during adulthood [23]. Interest-

ingly, Rb loss of function is highly prevalent in adrenal

pheochromocytomas [24, 25] and chimeric mice generated

from Rb-deficient cells exhibit hyperplasia of the AM [26].

Synthesis of adrenal steroid hormones (occurring in the

three layers of the AC) relies on cholesterol availability.

Cholesterol is thought to be de novo synthesized in neonatal

AGs. In contrast, circulating cholesterol becomes the main

substrate for hormone production during adulthood [27]. In

agreement with these facts, fatty acid binding proteins,

which regulate cholesterol synthesis, appear to be highly

expressed in neonatal in comparison with adult AGs (see

Fig. 2 and Table 1).

The coordinated metabolic response to AG maturation

illustrated by our proteomic study is schematically repre-

sented in Fig. 4. In AGs, a high glucose-6-phosphate-dehy-

drogenase activity has been reported [28] and a recent study

has highlighted the importance of phosphogluconate dehy-

drogenase (PGD) activity in AG reduced nicotinamide

adenine dinucleotide phosphate (NADPH) production [29].

PGD is up-regulated in adult AGs (Tables 1 and Fig. 4) and,

in parallel, we have also observed an increase in the relative

amounts of several metabolic enzymes. Diverse enzymes

*

*

*

*

**

*

Figure 4. AG metabolic reorganization during

postnatal development. Schematic repre-

sentation of metabolic enzymes with changes

in expression during AG postnatal develop-

ment. Proteins with a higher abundance in

adult AGs are represented in black. Proteins

with a higher level of expression in neonatal

AGs are shown in gray. Enzymes where

distinct subunits behave differentially regard-

ing postnatal expression are represented

as a black lettering inside a gray square.

Gray asterisk represents a NADPH producing

enzyme and black asterisks indicate

NADH producing sites. 1. Glyceraldehyde

3-phosphate dehydrogenase (E.C.: 1.2.1.12),

2. Phosphoglycerate kinase (E.C.: 2.7.2.3),

3. Phosphoglycerate mutase (E.C.: 5.4.2.1), 4.

Enolase (E.C.: 4.2.1.11), 5. Pyruvate dehy-

drogenase (E.C.: 1.2.4.1), 6. Dihydrolipoamide

dehydrogenase (E.C.: 1.8.1.4), 7. Isocitrate

dehydrogenase (E.C.: 1.1.1.41), 8. Oxogluta-

rate dehydrogenase (E.C.: 1.2.4.2), 9. Dihy-

drolipoamide S-succinyltransferase (E.C.:

2.3.1.61), 10. Fumarate hydratase (E.C.:

4.2.1.2), 11. Isovaleryl-CoA dehydrogenase

(E.C.: 1.3.99.10), 12. Methylcrotonoyl-CoA

carboxylase (E.C.: 6.4.1.4), 13. 3-oxoacid CoA-

transferase (E.C.: 2.8.3.5), 14. Enoyl-CoA

hydratase (E.C.: 4.2.1.17), 15. 3-hydroxyacyl-

CoA dehydrogenase (E.C.: 1.1.1.35), 16.

Propionyl-CoA carboxylase (E.C.: 6.4.1.3), 17.

Methylmalonyl-CoA mutase (RGD1564912)

(E.C.: 5.4.99.2), 18. Glutamate oxaloacetate

transaminase (E.C.: 2.6.1.1), 19. Glucosamine-

6-phosphate isomerase (E.C.: 3.5.99.6), 20.

Glycerol-3-phosphate dehydrogenase (E.C.:

1.1.1.8), 21. Phosphogluconate dehy-

drogenase (decarboxylating) (E.C.: 1.1.1.44),

22. 2-hydroxyglutarate dehydrogenase

(RGD1307976) (E.C.: 1.1.99.2), 23. Pyruvate

dehydrogenase kinase 1 (E.C.: 2.7.11.2).



participating in glycolysis, citric acid cycle, as well as in

leucine, valine, isoleucine, and propionate catabolic path-

ways were up-regulated (�two-fold increase) with AG

maturation, although we also found some enzymes with

higher expression level in neonatal glands (Figs. 2 and 4 and

Table 1). Many of the enzymatic activities up-regulated with

AG ontogeny are directly implicated in NAD(P)H produc-

tion (Fig. 4, asterisks). Biosynthesis of AG steroid hormones

requires a high supply of NADPH for hydroxylation of

cholesterol and its C21 derivatives. These reactions, includ-

ing cholesterol side-chain cleavage [30], depend essentially

on NADPH that is generated by mitochondrial transhy-

drogenases at the expense of NADH [30, 31]. The cyto-

plasmic NADPH, utilized for 21-hydroxylation, originates

from the pentose phosphate pathway. In addition, NADH is

required for correct enzymatic detoxification of isoca-

proaldehyde, a product of side-chain cleavage of cholesterol

generated during the initial step of steroidogenesis [32]. In

parallel to the increase in reduced molecules production, an

up-regulation in the amount of ferredoxin reductase was

observed. This enzymatic activity is required for transferring

electrons from NADPH to the mitochondrial P450 enzymes

involved in steroid synthesis. The overall picture emerging

from our proteomic study points to a metabolic adaptation

leading to an increased production of reducing molecules in

matured AC, using mainly glucose and amino acids as

precursors. The increase in adult AGs of the leucyl- and

glutamyl-prolyl-tRNA synthetase enzymes suggests that the

accelerated catabolism of amino acid could be compensated

with an optimization of protein translation by up-regulation

of specific tRNA synthetases.

In conclusion, the results presented here constitute the

first proteomic analysis performed so far of postnatal rodent

AG development. We have identified molecular changes

that are relevant for understanding AG development as well

as modifications in pathophysiological conditions. Further

studies will be required to describe in detail the role of the

identified proteins in AG maturation and in the specific

functions of neonatal and adult AGs.

Support was obtained from the Juan March Foundation, theMarcelino Botın Foundation, the Spanish Ministry of Scienceand Education, the Spanish Ministry of Health (TERCEL), andthe Andalusian Government. CIBERNED is funded by theInstituto de Salud Carlos III.

The authors have declared no conflict of interest.

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differential proteomic analysis of adrenal gland during postnatal development

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