A Human Variant of Glucose-Regulated Protein 94 That Inefficiently Supports IGF Production.

IGFs are critical for normal intrauterine and childhood growth and sustaining health throughout life. We showed previously that the production of IGF-1 and IGF-2 requires interaction with the chaperone glucose-regulated protein 94 (GRP94) and that the amount of secreted IGFs is proportional to the GRP94 activity. Therefore, we tested the hypothesis that functional polymorphisms of human GRP94 affect IGF production and thereby human health. We describe a hypomorphic variant of human GRP94, P300L, whose heterozygous carriers have 9% lower circulating IGF-1 concentration. P300L was found first in a child with primary IGF deficiency and was later shown to be a noncommon single-nucleotide polymorphism with frequencies of 1%-4% in various populations. When tested in the grp94(-/-) cell-based complementation assay, P300L supported only approximately 58% of IGF secretion relative to wild-type GRP94. Furthermore, recombinant P300L showed impaired nucleotide binding activity. These in vitro data strongly support a causal relationship between the GRP94 variant and the decreased concentration of circulating IGF-1, as observed in human carriers of P300L. Thus, mutations in GRP94 that affect its IGF chaperone activity represent a novel causal genetic mechanism that limits IGF biosynthesis, quite a distinct mechanism from the known genes in the GH/IGF signaling network.

Effects of sustained exercise on GH-IGFs axis in gilthead sea bream (Sparus aurata).

The endocrine system regulates growth mainly through the growth hormone (GH)/insulin-like growth factors (IGFs) axis and, although exercise promotes growth, little is known about its modulation of these factors. The aim of this work was to characterize the effects of 5 wk of moderate sustained swimming on the GH-IGFs axis in gilthead sea bream fingerlings. Plasma IGF-I/GH ratio and tissue gene expression of total IGF-I and three splice variants, IGF-II, three IGF binding proteins, two GH receptors, two IGF-I receptors, and the downstream molecules were analyzed. Fish under exercise (EX) grew more than control fish (CT), had a higher plasma IGF-I/GH ratio, and showed increased hepatic IGF-I expression (mainly IGF-Ia). Total IGF-I expression levels were similar in the anterior and caudal muscles; however, IGF-Ic expression increased with exercise, suggesting that this splice variant may be the most sensitive to mechanical action. Moreover, IGFBP-5b and IGF-II increased in the anterior and caudal muscles, respectively, supporting enhanced muscle growth. Furthermore, in EX fish, hepatic IGF-IRb was reduced together with both GHRs; GHR-II was also reduced in anterior muscle, while GHR-I showed higher expression in the two muscle regions, indicating tissue-dependent differences and responses to exercise. Exercise also increased gene and protein expression of target of rapamycin (TOR), suggesting enhanced muscle protein synthesis. Altogether, these data demonstrate that moderate sustained activity may be used to increase the plasma IGF-I/GH ratio and to potentiate growth in farmed gilthead sea bream, modulating the gene expression of different members of the GH-IGFs axis (i.e., IGF-Ic, IGF-II, IGFBP-5b, GHR-I, and TOR).

The Obesity-Linked Gene Nudt3 Drosophila Homolog Aps Is Associated With Insulin Signaling.

Several genome-wide association studies have linked the Nudix hydrolase family member nucleoside diphosphate-linked moiety X motif 3 (NUDT3) to obesity. However, the manner of NUDT3 involvement in obesity is unknown, and NUDT3 expression, regulation, and signaling in the central nervous system has not been studied. We performed an extensive expression analysis in mice, as well as knocked down the Drosophila NUDT3 homolog Aps in the nervous system, to determine its effect on metabolism. Detailed in situ hybridization studies in the mouse brain revealed abundant Nudt3 mRNA and protein expression throughout the brain, including reward- and feeding-related regions of the hypothalamus and amygdala, whereas Nudt3 mRNA expression was significantly up-regulated in the hypothalamus and brainstem of food-deprived mice. Knocking down Aps in the Drosophila central nervous system, or a subset of median neurosecretory cells, known as the insulin-producing cells (IPCs), induces hyperinsulinemia-like phenotypes, including a decrease in circulating trehalose levels as well as significantly decreasing all carbohydrate levels under starvation conditions. Moreover, lowering Aps IPC expression leads to a decreased ability to recruit these lipids during starvation. Also, loss of neuronal Aps expression caused a starvation susceptibility phenotype while inducing hyperphagia. Finally, the loss of IPC Aps lowered the expression of Akh, Ilp6, and Ilp3, genes known to be inhibited by insulin signaling. These results point toward a role for this gene in the regulation of insulin signaling, which could explain the robust association with obesity in humans.

Interleukin-driven insulin-like growth factor promotes prostatic inflammatory hyperplasia.

Prostatic inflammation is of considerable importance to urologic research because of its association with benign prostatic hyperplasia and prostate cancer. However, the mechanisms by which inflammation leads to proliferation and growth remain obscure. Here, we show that insulin-like growth factors (IGFs), previously known as critical developmental growth factors during prostate organogenesis, are induced by inflammation as part of the proliferative recovery to inflammation. Using genetic models and in vivo IGF receptor blockade, we demonstrate that the hyperplastic response to inflammation depends on interleukin-1-driven IGF signaling. We show that human prostatic hyperplasia is associated with IGF pathway activation specifically localized to foci of inflammation. This demonstrates that mechanisms of inflammation-induced epithelial proliferation and hyperplasia involve the induction of developmental growth factors, further establishing a link between inflammatory and developmental signals and providing a mechanistic basis for the management of proliferative diseases by IGF pathway modulation.

Lowered insulin signalling ameliorates age-related sleep fragmentation in Drosophila.

Sleep fragmentation, particularly reduced and interrupted night sleep, impairs the quality of life of older people. Strikingly similar declines in sleep quality are seen during ageing in laboratory animals, including the fruit fly Drosophila. We investigated whether reduced activity of the nutrient- and stress-sensing insulin/insulin-like growth factor (IIS)/TOR signalling network, which ameliorates ageing in diverse organisms, could rescue the sleep fragmentation of ageing Drosophila. Lowered IIS/TOR network activity improved sleep quality, with increased night sleep and day activity and reduced sleep fragmentation. Reduced TOR activity, even when started for the first time late in life, improved sleep quality. The effects of reduced IIS/TOR network activity on day and night phenotypes were mediated through distinct mechanisms: Day activity was induced by adipokinetic hormone, dFOXO, and enhanced octopaminergic signalling. In contrast, night sleep duration and consolidation were dependent on reduced S6K and dopaminergic signalling. Our findings highlight the importance of different IIS/TOR components as potential therapeutic targets for pharmacological treatment of age-related sleep fragmentation in humans.

Identification of microRNA-18a as a novel regulator of the insulin-like growth factor-1 in the proliferation and regeneration of deer antler.

To investigate the effect of miR-18a on the regulation of the insulin-like growth factor (IGF-1) during growth of antlers in sika deer, miRNA Chip analysis, Target Scan and real-time PCR analysis were used to identify miRNAs that bind to the 3'-UTR of IGF-1. An miR-18a mimic was transfected into antler cartilage cells and the expression levels were quantified by real-time PCR. Dual luciferase assays revealed that miR-18a binds to the 3'-UTR of the IGF-1 gene thus indicating this to be a target gene regulated by miR-18a. MTT assays and cell-cycle analyses confirmed that miR-18a significantly inhibited proliferation of cartilage cells. In contrast, transfection of miR-18a inhibitors increased proliferation. Furthermore, Western blot analysis showed that over-expression of miR-18a down-regulated IGF-1 protein levels while IGF-1 expression was increased after transfection of miR-18a inhibitors. Thus, miR-1 down-regulated IGF-1 expression thus implicating miR-18a as an important regulator of antler proliferation.

Intramammary infusion of Panax ginseng extract in the bovine mammary gland at cessation of milking modifies components of the insulin-like growth factor system during involution.

The objective of this study was to evaluate the effects of a single intramammary infusion of Panax ginseng extract (GS) on insulin-like growth factors (IGF) in bovine mammary gland during early involution. Eight mammary quarters from six nonpregnant cows in late lactation were infused with 10 mL of ginseng extract solution (3 mg/mL), six quarters were treated with 10 mL of placebo (vehicle alone) and six quarters were maintained as uninoculated controls. Milking was interrupted after infusion. Concentrations of IGF1 in mammary secretions were higher in GS-treated quarters than in placebo and uninoculated control quarters at 24, 48 and 72 h post-treatment (p<0.05). Treatment with GS did not affect mammary secretion of IGF2 (p=0.942). At 7 d of post-lactational involution, a decrease of immunostained area and mRNA expression for IGF1 was observed in mammary tissue of GS-treated quarters compared with placebo-treated quarters and uninoculated controls (p<0.05). The IGF2 immunostained area and mRNA expression for this growth factor were not affected by GS treatment (p=0.216 and p=0.785, respectively). An increase in protein levels and mRNA expression in mammary tissue of IGFBP3, IGFBP4 and IGFBP5 was observed in GS-treated quarters compared with placebo-treated quarters and uninoculated controls (p<0.05). These results provide evidence that intramammary inoculation of GS extract at cessation of milking may promote early mammary involution through the inhibition of IGF1 local production and bioavailability.

A progestin (17α,20ß-dihydroxy-4-pregnen-3-one) stimulates early stages of spermatogenesis in zebrafish.

Recently, evidence has been provided for multiple regulatory functions of progestins during the late mitotic and meiotic phases of spermatogenesis in teleost fish. For example, our previous studies suggested that 17α,20ß-dihydroxy-4-pregnen-3-one (DHP), potentially via Sertoli cells that express the progesterone receptor (pgr) gene, can contribute to the regulation of zebrafish spermatogenesis. To further our understanding of the function of DHP at early spermatogenetic stages, we investigated in the present study the expression of genes reflecting Sertoli cell function and spermatogenic development in adult zebrafish testis after DHP treatment in tissue culture. Moreover, using an in vivo model of estrogen-mediated down-regulation of androgen production to interrupt adult spermatogenesis, we studied the effects of DHP on estrogen-interrupted spermatogenesis. In this model, DHP treatment doubled the testis weight, and all differentiating germ cell types, such as type B spermatogonia and primary spermatocytes, were abundantly present and incorporated the DNA-synthesis marker (BrdU). Accordingly, transcript levels of germ cell marker genes were up-regulated. Moreover, transcripts of two Sertoli cell-derived genes anti-müllerian hormone (amh) and gonadal soma-derived growth factor (gsdf) were up-regulated, as were three genes of the insulin-like growth factor signaling system, insulin-like growth factor 2b (igf2b), insulin-like growth factor 3 (igf3) and insulin-like growth factor 1b receptor (igf1rb). We further analyzed the relationship between these genes and DHP treatment using a primary zebrafish testis tissue culture system. In the presence of DHP, only igf1rb mRNA levels showed a significant increase among the somatic genes tested, and germ cell marker transcripts were again up-regulated. Taken together, our results show that DHP treatment induced the proliferation of early spermatogonia, their differentiation into late spermatogonia and spermatocytes as well as expression of marker genes for these germ cell stages. DHP-mediated stimulation of spermatogenesis and hence growth of spermatogenic cysts and the associated increase in Sertoli cell number may in part explain the elevated expression of Sertoli cell genes, but our data also suggest an up-regulation of the activity of the Igf signaling system.

Interplay between autophagy and apoptosis in the development of Danio rerio follicles and the effects of a probiotic.

The present study investigated autophagic processes in Danio rerio preovulatory follicles (Stage III and IV). There were more autophagosomes, as revealed by electron microscopy, in follicles from females fed the probiotic Lactobacillus rhamnosus IMC 501. This was confirmed by increased expression of genes involved in the autophagic process, namely ambra1, becn1, lc3 and uvrag. In addition, preovulatory follicles from females fed the probiotic contained more microtubule-associated protein 1 light chain 3 isoform II (LC3-II) and less p62 protein. The increased autophagy in preovulatory follicles from females fed the probiotic was concomitant with a decrease in the apoptotic process in the ovary, as evidenced by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling analysis and confirmed by lower expression of genes involved in apoptosis (i.e., p53, bax, apaf and cas3) and higher expression as igfII and igf1r. The results of the present study provide preliminary evidence of the involvement of autophagy during follicle development in the zebrafish ovary. In addition, we have demonstrated for the first time that a functional food, such as L. rhamnosus IMC 501, can modulate the balance between apoptosis and autophagy that regulates ovary physiology in zebrafish by inhibiting follicular apoptosis and improving follicular survival.

Deletion of muscle GRP94 impairs both muscle and body growth by inhibiting local IGF production.

Insulin-like growth factors (IGFs) are critical for development and growth of skeletal muscles, but because several tissues produce IGFs, it is not clear which source is necessary or sufficient for muscle growth. Because it is critical for production of both IGF-I and IGF-II, we ablated glucose-regulated protein 94 (GRP94) in murine striated muscle to test the necessity of local IGFs for normal muscle growth. These mice exhibited smaller skeletal muscles with diminished IGF contents but with normal contractile function and no apparent endoplasmic reticulum stress response. This result shows that muscles rely on GRP94 primarily to support local production of IGFs, a pool that is necessary for normal muscle growth. In addition, body weights were ∼30% smaller than those of littermate controls, and circulating IGF-I also decreased significantly, yet glucose homeostasis was maintained with little disruption to the growth hormone pathway. The growth defect was complemented on administration of recombinant IGF-I. Thus, unlike liver production of IGF-I, muscle IGF-I is necessary not only locally but also globally for whole-body growth.

Insulin-like growth factors in endometrioid adenocarcinoma: correlation with clinico-pathological features and estrogen receptor expression.

BACKGROUND: Endometrial carcinoma is a common malignancy of female genital tract. Insulin-like growth factor is known to elicit estrogen-induced mitogenic activity and anti-apoptotic effect in endometrial tissues. METHODS: The retrospective study investigated the expression of insulin-like growth factors, estrogen receptors and their associations in endometrioid adenocarcinoma (EAC) from 80 EAC patients in immunohistochemistry, and 58 EAC patients and 42 control patients in quantitative RT-PCR. The Pearson correlation analysis was used to analyze their correlations with clinic-pathological parameters. RESULTS: Our results showed that insulin-like growth factor-1 and insulin-like growth factor-2 mRNA levels were higher in tumor tissues and tumor-adjacent tissues than those in control cells, and were inversely correlated with the malignancy of the tumor with a positive correlation with ERα and ERß expression. Insulin-like growth factor-1R protein expression was correlated with clinical stage, and insulin-like growth factor-2R protein expression was inversely correlated with histological grade. CONCLUSIONS: Insulin-like growth factor system plays an important role in estrogen-induced endometrial carcinogenesis, and overexpression of insulin-like growth factor-1R in the advanced endometrioid adenocarcinoma is not estrogen-dependent.

Insulin-like growth factor receptor expression is associated with aggressive phenotypes and has therapeutic activity in biliary tract cancers.

Insulin-like growth factor (IGF)-I receptor (IGF-IR) signaling is required for carcinogenicity and progression of several cancers but the function of this pathway and its utility as a therapeutic target have not been studied comprehensively in biliary tract carcinomas (BTC). We investigated the immunohistochemical expression of elements of the IGF axis, matrilysin, overexpression of p53 and the methylation status of the IGFBP-3 promoter in 80 surgically resected BTC. We also assessed the effect of IGF-IR blockade on signal transduction, proliferation and survival in three BTC cell lines using a new tyrosine kinase inhibitor, BMS-536924, and dominant negative IGF-IR (IGF-IR/dn). The effects of IGF-IR blockade was also studied in nude mouse xenograft models. IGF-I was expressed in 60% and IGF-II in 50% of tumors. High expression was associated with tumor size. IGF-IR was expressed in 69% of the cases and was associated with advanced stage and matrilysin expression. Hypermethylation of the IGFBP-3 promoter was detected in 41% of BTC and was inversely correlated with p53 expression. BMS-536924 blocked autophosphorylation of IGF-IR and both Akt and ERK activation by both IGF-I and insulin. BMS-536924 suppressed proliferation and tumorigenicity in vitro in a dose-dependent fashion. This inhibitor upregulated chemotherapy-induced apoptosis in a dose-dependent fashion. Moreover, IGF-IR blockade was effective against tumors in mice. IGF-IR might identify a subset of BTC with a particularly aggressive phenotype and is a candidate therapeutic target in this disease. BMS-536924 might have significant therapeutic utility.

Gene expression in swine granulosa cells and ovarian tissue during the estrous cycle.

The components of the insulin-like growth factor (IGF) system appear to be involved in regulation of ovarian follicular growth and atresia in the pig. We investigated the expression pattern of mRNAs for IGF1 (IGF1), its binding proteins (IGFBP1, IGFBP2, IGFBP3, and IGFBP5), and epidermal growth factor in swine follicle cells and ovarian tissue throughout the estrous cycle using the real-time quantitative PCR technique. The results of gene expression were analyzed using linear regression with gene expression as a dependent variable and days of estrous cycle as an independent variable. Additionally, an analysis was made of the correlation of expression levels with plasma concentration of follicle-stimulating hormone, luteinizing hormone, estradiol-17ß, progesterone, and prolactin. Expression of mRNA of all of these genes was detected in granulosa cells and ovarian tissue. IGFBP3 mRNA showed a quadratic expression pattern (P ≤ 0.001) and was significantly and positively correlated with progesterone (r = 0.81; P ≤ 0.01) but negatively correlated with prolactin (r = -0.596; P ≤ 0.05). Expression of the other genes was unaffected by the stage of the estrous cycle. Real-time quantitative PCR effectively detected all transcripts, including the very low levels of IGFBP1 transcripts, and could be used for studies of follicle dynamics.

Differential granulosa cell gene expression in young women with diminished ovarian reserve.

OBJECTIVE: To investigate if a diagnosis of diminished ovarian reserve (DOR) is associated with a differential gene profile of ovarian granulosa cells (GCs) in infertile women undergoing in vitro fertilization (IVF). DESIGN: Prospective Cohort Study. SETTING: Academic IVF Program. PATIENTS: Infertile women <38 years were prospectively enrolled into 2 groups: normal ovarian reserve (NOR, follicle-stimulating hormone [FSH] < 10 mIU/mL, n = 4) and DOR (FSH ≥ 10.0 mIU/mL, n = 4). INTERVENTIONS: Cumulus (C) and mural (M) GCs were isolated at egg retrieval; messenger RNA was extracted and transcribed. MAIN OUTCOME MEASURE(S): Differential gene expression in cerebellar granule cells (CGCs) in the 2 groups was assessed by cDNA microarray. Microarray findings were validated by quantitative real-time polymerase chain reaction (qRTPCR) in CGCs and explored in multinucleated giant cells (MGCs). RESULTS: Of the 1256 differentially regulated genes identified in CGCs of women with DOR, the insulin-like growth factor (IGF) family was a biologically relevant gene family of a priori interest. Downregulation of IGF1 and IGF2 ligands (-3.28- and -2.54-fold, respectively), and their receptors, (-3.53- and -1.32-fold downregulation of IGF1R and IGF2R, respectively) was identified in luteinized CGCs in women with DOR compared to those with NOR. Downregulation of both IGF1 and IGF 2 ligands (-4.35- and 3.89-fold, respectively) was furthermore observed in MGCs in women with DOR compared to those with NOR; no differences in the expression of respective receptors were however observed in MGCs in the 2 groups. CONCLUSIONS: Components of the IGF gene family are downregulated in GCs of women with DOR. These findings maybe contributory to the reproductive compromise observed in women with DOR, and merit further exploration.

Cells producing insulin-like androgenic gland hormone of the giant freshwater prawn, Macrobrachium rosenbergii, proliferate following bilateral eyestalk-ablation.

We found that the androgenic gland (AG) of Macrobrachium rosenbergii possesses three cell types. Type I cells are small polygonal shaped-cells (13.4 µm in diameter), stain strongly with hematoxylin-eosin (H&E), have abundant multilayered rough endoplasmic reticulum (rER), and nuclei containing mostly heterochromatin. Type II cells are slightly larger (18.6 µm in diameter), stain lightly with H&E, have rER with dilated cisternae, and nuclei containing mostly euchromatin. Type III cells (previously undescribed) are similar in size and shape to type I cells, but the cytoplasm is unstained and they have a high amount of smooth endoplasmic reticulum (sER) and mitochondria with tubular cristae. Bilateral eyestalk-ablation resulted in AG hypertrophy with a proliferation and predominance of type I cells as determined by bromodeoxyuridine (BrdU) assays. Expression of insulin-like androgenic gland hormone (Mr-IAG), determined by immunohistochemistry, was weak in type I cells, strong in type II cells of both the intact and eyestalk-ablated, and negative in type III cells. It was also detected in spermatogonia, nurse cells, and epithelium lining of the spermatic duct. The function of Mr-IAG in these tissues is yet to be elucidated but the distribution implies a strong role in male reproduction.

Zebrafish eggs used as bioreactors for the production of bioactive tilapia insulin-like growth factors.

Multiple advantages-including the short generation time, large numbers of fertilized eggs, low cost of cultivation and easy maintenance favor the use of fish as bioreactors for the production of pharmaceutical proteins. In the present study, zebrafish eggs were used as bioreactors to produce mature tilapia insulin-like growth factors (IGFs) proteins using the oocyte-specific zona pellucida (zp3) promoter. The chimeric expression plasmids, pT2-ZP-tIGFs-IRES-hrGFP, in which hrGFP was used as reporter of tilapia IGFs expression, were designed to established Tg (ZP:tIGFs:hrGFP) transgenic lines for the expression of tilapia IGF-1 and IGF-2. Recombinant tilapia IGF-1 and IGF-2 were expressed as soluble forms in cytoplasm of fertilized eggs. The content level of tilapia IGF-1 and IGF-2 were 6.5 and 5.0% of the soluble protein, respectively. Using a simple Ni-NTA affinity chromatography purification process, 0.58 and 0.49 mg of purified tilapia IGF-1 and IGF-2 were obtained, respectively, from 650 fertilized eggs. The biological activity of the purified tilapia IGF-1 and IGF-2 was confirmed via a colorimetric bioassay to monitor the growth stimulation of zebrafish embryonic cells (ZF4), tilapia ovary cells (TO-2) and human osteosarcoma epithelial cells (U2OS). These results demonstrate that the use of zebrafish eggs as bioreactors is a promising approach for the production of biological recombinant proteins.

Elevated epithelial insulin-like growth factor expression is a risk factor for lung cancer development.

Insulin-like growth factor (IGF)-I receptor (IGF-IR) signaling has been implicated in several human neoplasms. However, the role of serum levels of IGFs in lung cancer risk is controversial. We assessed the role of tissue-derived IGFs in lung carcinogenesis. We found that IGF-I and IGF-II levels in bronchial tissue specimens containing high-grade dysplasia were significantly higher than in those containing normal epithelium, hyperplasia, and squamous metaplasia. Derivatives of human bronchial epithelial cell lines with activation mutation in KRAS(V12) or loss of p53 overexpressed IGF-I and IGF-II. The transformed characteristics of these cells were significantly suppressed by inactivation of IGF-IR or inhibition of IGF-I or IGF-II expression but enhanced by overexpression of IGF-IR or exposure to the tobacco carcinogens (TC) 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone and benzo(a)pyrene. We further determined the role of IGF-IR signaling in lung tumorigenesis by determining the antitumor activities of the selective IGF-IR tyrosine kinase inhibitor cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl]-1-(2-phenyl-quinolin-7-yl)-imidazo [1,5-a]pyrazin-8-ylamine using an in vitro progressive cell system and an in vivo mouse model with a lung-specific IGF-I transgene after exposure to TCs, including 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone plus benzo(a)pyrene. Our results show that airway epithelial cells produce IGFs in an autocrine or paracrine manner, and these IGFs act jointly with TCs to enhance lung carcinogenesis. Furthermore, the use of selective IGF-IR inhibitors may be a rational approach to controlling lung cancer.

Expression of the IGF and the aromatase/estrogen receptor systems in human adrenal tissues from early infancy to late puberty: implications for the development of adrenarche.

Adrenarche is a process of postnatal sexual maturation occurring in higher primates, in which there is an increase in the secretion of adrenal androgens. It is the consequence of a process of postnatal organogenesis characterized by the development of a new zone in the adrenal cortex, the zona reticularis (ZR). The mechanism of this phenomenon remains poorly understood, suggesting that it might be a multifactorial event. A relationship between circulating IGF-I, insulin sensitivity, and adrenal androgens has been postulated. Boys and girls have different patterns of changes in insulin sensitivity at puberty, perhaps secondary to differences in the estrogen milieu. Estrogen effects may also play a role in premature adrenarche. Peripheral or local IGF-1 actions could regulate adrenal progenitor cell proliferation and migration. Since adrenal progenitor cells as well as IGF-I and the IGF-R1 are located in the outer zone of the adrenal cortex during childhood and adolescence, this peripheral cell layer, below the capsule, may contain undifferentiated progenitor cells. Therefore, the IGF-R1 signaling pathway might positively modulate the proliferation and migration of adrenal progenitor cell to stimulate the development of adrenal zones, including ZR. However, no evidence of a direct action of IGF-I on ZR was found. In addition, a role for estrogens in the ontogenesis of ZR is suggested by the presence of aromatase (CYP19) in the subcapsular zona glomerulosa and in the adrenal medulla. Estrogens produced locally could act on ZR by interacting with estrogen receptor beta (ERbeta), but not alpha, and membrane estrogen receptor GPR-30. An estradiol-induced increase in DHEA/cortisol ratio was indeed seen in cultures of adrenocortical cells from post-adrenarche adrenals. In summary, several lines of evidence point to the action of multiple factors, such as local adrenal maturational changes and peripheral metabolic signals, on postnatal human adrenal gland ZR formation.