AINO MÄNTYSELKÄ
ADRENARCHE
Dissertations in Health Sciences
PUBLICATIONS OF
THE UNIVERSITY OF EASTERN FINLAND
A D R E N A R C H E
PREDISPOSING FACTORS, PRESENTATION AND EFFECTS ON HEALTH
Aino Mäntyselkä
A D R E N A R C H E
PREDISPOSING FACTORS, PRESENTATION AND EFFECTS ON HEALTH
Publications of the University of Eastern Finland Dissertations in Health Sciences
No 593
University of Eastern Finland Kuopio
2020
To be presented by permission of the
Faculty of Health Sciences, University of Eastern Finland for public examination in Auditorium CA 101, Canthia, Kuopio
on Friday, October 30th, 2020, at 12 o’clock noon
Series Editors
Professor Tomi Laitinen, M.D., Ph.D.
Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences
Associate professor (Tenure Track) Tarja Kvist, Ph.D.
Department of Nursing Science Faculty of Health Sciences Professor Ville Leinonen, M.D., Ph.D.
Institute of Clinical Medicine, Neurosurgery Faculty of Health Sciences
Associate Professor (Tenure Track) Tarja Malm, Ph.D.
A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences
Lecturer Veli-Pekka Ranta, Ph.D.
School of Pharmacy Faculty of Health Sciences
Distributor:
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P.O.Box 1627 FI-70211 Kuopio, Finland
www.uef.fi/kirjasto
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Grano oy Jyväskylä, 2020
ISBN: 978—952-61-3595-3 (print/nid.) ISBN: 978-952-61-3596-0 (PDF)
ISSNL: 1798-5706 ISSN: 1798-5706 ISSN: 1798-5714 (PDF)
Author’s address: Department of Pediatrics Kuopio University Hospital University of Eastern Finland KUOPIO
FINLAND
Doctoral programme: Doctoral Programme of Clinical Research Supervisors: Professor Jarmo Jääskeläinen, M.D., Ph.D.
Department of Pediatrics, School of Medicine University of Eastern Finland
KUOPIO FINLAND
Professor Timo Lakka, M.D., Ph.D.
Institute of Biomedicine/Physiology, School of Medicine University of Eastern Finland
KUOPIO FINLAND
Docent Virpi Lindi, Ph.D. †
Institute of Biomedicine/Physiology, School of Medicine University of Eastern Finland
KUOPIO FINLAND
Reviewers: Docent Marja Ojaniemi, M.D., Ph.D.
Department of Children and Adolescents University of Oulu
OULU FINLAND
Docent Matti Hero, M.D., Ph.D.
Children’s Hospital
Helsinki University Hospital HELSINKI
FINLAND
Opponent: Docent Päivi Keskinen, M.D, Ph.D.
Department of Pediatrics Tampere University Hospital TAMPERE
FINLAND
Mäntyselkä, Aino
Adrenarche. Predisposing factors, presentation and effects on health Kuopio: University of Eastern Finland
Publications of the University of Eastern Finland Dissertations in Health Sciences 593. 2020, 138 p.
ISBN: 978-952-61-3595-3 (print) ISSNL: 1798-5706
ISSN: 1798-5706
ISBN: 978-952-61-3596-0 (PDF) ISSN: 1798-5714 (PDF)
ABSTRACT
Adrenarche means an increase in the production of adrenal androgens in childhood.
Previous studies have focused mostly on premature adrenarche, which has been linked to insulin resistance, especially in overweight children. The regulation of adrenarche is scarcely known, and its timing and intensity vary among inviduals.
Adrenarche has not earlier been studied much in general populations of children.
Thus, the associations of timing and intensity of adrenarche with the health of children are not known.
Adrenarche occurs some years before the onset of puberty. An increase in serum androgen levels can lead to the clinical signs of adrenarche, including adult-type body odor, oily hair and skin, acne, comedones, as well as axillary and pubic hair.
Many children with adrenarche also have accelerated growth. A serum dehydroepiandrosterone sulfate (DHEAS) concentration of 1 µmol/L has been considered the cut-off level for biochemical adrenarche, particularly in research.
The purpose of this doctoral thesis was to study the presentation of adrenarche in girls and in boys, the associations of serum DHEAS with adiposity and other cardiometabolic risk factors, the associations of lifestyle factors, such as diet, physical activity and sedentary behavior with serum DHEAS and insulin-like growth factor 1 (IGF-1), and the associations of serum DHEAS and IGF-1 with cognition. The study population consisted of 437 prepubertal children aged 6-8 years participating in the Physical activity and Nutrition in Children (PANIC) study.
The clinical signs of adrenarche were more common in girls than in boys, but biochemical adrenarche without clinical signs was more common in boys. This difference between genders was probably due to a higher body fat content and more potent peripheral androgen conversion in girls. The prevalence of premature adrenarche was 8.6% in girls and 1.8% in boys. Higher serum DHEAS was associated with lower plasma low-density lipoprotein (LDL) cholesterol after adjustment for birth weight and current body mass index. The previously found association of increased cardiometabolic risk and premature adrenarche may be due to
confounding factors such as low birth weight and excess weight rather than premature adrenarche. Lifestyle factors had weak or moderate associations with serum DHEAS and IGF-1 concentrations. For example, consumption of low-fiber grain products was positively associated with DHEAS and energy intake was positively associated with IGF-1. Vigorous physical activity was inversely associated with DHEAS and total sedentary behavior was positively associated with IGF-1.
Serum DHEAS was not associated with cognition, but higher serum IGF-1 was related to better cognition among boys.
This doctoral thesis provides new evidence on the prevalence and phenotype of physiological adrenarche and its effects in normal population.
National Library of Medicine Classification: WS 450, WS 440, WK 755, QU 107
Medical Subject Headings: Androgens/metabolism; Adrenarche; Child; Sex Characteristics;
Body Composition; Insulin-Like Growth Factor I; Dehydroepiandrosterone Sulfate;
Cardiovascular Diseases; Risk Factors; Cognition; Exercise; Diet Surveys;
Finland/epidemiology
Mäntyselkä, Aino
Adrenarche. Predisposing factors, presentation and effects on health Kuopio: Itä-Suomen yliopisto
Publications of the University of Eastern Finland Dissertations in Health Sciences 593. 2020, 138 s.
ISBN: 978-952-61-3595-3(nid.) ISSNL: 1798-5706
ISSN: 1798-5706
ISBN: 978-952-61-3596-0 (PDF) ISSN: 1798-5714(PDF)
TIIVISTELMÄ
Adrenarke tarkoittaa lisämunuaiskuoren miessukupuolihormonien tuotannon käynnistymistä lapsuusiässä. Aikaisemmin on tutkittu erityisesti ennenaikaista adrenarkea, mihin on liittynyt insuliiniresistenssiä varsinkin ylipainoisilla lapsilla.
Adrenarken säätely tunnetaan huonosti ja sen ajoitus ja voimakkuus vaihtelevat huomattavasti yksilöiden välillä. Adrenarkea ei ole aiemmin juurikaan tutkittu aiemmin tavallisista lapsista koostuvissa populaatioissa. Siten ei tiedetä adrenarken ajoituksen ja voimakkuuden yhteyksiä lasten terveyteen. Lisämunuaiskuoren androgeenituotannon käynnistyminen tapahtuu tavallisesti joitakin vuosia ennen varsinaisen murrosiän alkua. Lisääntynyt seerumin androgeenien pitoisuus voi johtaa kliinisiin löydöksiin kuten aikuistyyppiseen hienhajuun, hiusten ja ihon rasvoittumiseen, sekä aknen, comedojen, kainalokarvoituksen ja häpykarvoituksen ilmaantumiseen. Monella lapsella esiintyy myös pituuskasvun kiihtymistä.
Biokemiallisen adrenarken rajana lähinnä tutkimuksissa on pidetty seerumin dehydroepiandrosteroni (DHEAS) -pitoisuutta 1 µmol/L.
Tässä väitöskirjatyössä tutkittiin adrenarkea esimurrosikäisillä tytöillä ja pojilla.
Tavoitteena oli selvittää seerumin DHEAS-pitoisuuden yhteyttä kehon rasvakoostumukseen ja muihin sydän- ja verisuonisairauksien riskitekijöihin ja kuvata seerumin DHEAS- ja insuliininkaltaisen kasvutekijä 1:n (IGF-1) pitoisuuksiiin lapsuusiässä yhteydessä olevia tekijöitä, ennen kaikkea ravitsemuksen ja liikunnan yhteyttä. Lisäksi selvitettiin seerumin DHEAS- ja IGF-1- pitoisuuksien yhteyttä kognitioon. Tutkimusaineistona oli Lasten liikunta- ja ravitsemus-tutkimuksen (PANIC) aineiston (Itä-Suomen yliopisto, fysiologian yksikkö) 437 lasta 6-8 vuoden iässä.
Adrenarken kliiniset merkit olivat yleisempiä tytöillä kuin pojilla, mutta biokemiallinen adrenarke ilman kliinisiä merkkejä oli yleisempi pojilla. Ero sukupuolten välillä johtui todennäköisesti tyttöjen suuremmasta kehon rasvakoostumuksesta ja tehokkaammasta androgeenien perifeerisestä konversiosta.
Ennenaikaisen adrenarken prevalenssi oli tytöillä 8.6% ja pojilla 1.8%. Kun
huomioitiin lapsen syntymäkoko ja nykyinen painoindeksi, korkeampi DHEAS- pitoisuus oli yhteydessä matalampaan LDL-kolesterolipitoisuteen. Aikaisemmissa tutkimuksissa havaittu yhteys lisääntyneen sydän- ja verisuonisairauksien riskin ja ennenaikaisen adrenarken välillä voi johtua enemmän matalasta syntymäpainosta ja ylipainosta kuin ennenaikasesta adrenarkesta sinällään. Elämäntapatekijöillä oli vain heikkoja tai kohtalaisia yhteyksiä seerumin DHEAS- ja IGF-1 pitoisuuksiin.
Esimerkiksi suurempi vähäkuituisten viljatuotteiden saanti oli yhteydessä korkeampaan seerumin DHEAS-pitoisuuteen, ja suurempi energian saanti korkeampaan IGF-1-pitoisuuteen. Suurempi raskaan liikunnan määrä oli yhteydessä matalampaan DHEAS-pitoisuuteen ja suurempi fyysisen passiivisuuden määrä korkeampaan IGF-1-pitoisuuteen. Seerumin DHEAS-pitoisuus ei yhdistynyt parempaan kognitiiviseen suoriutumiseen, mutta korkeampi IGF-1-pitoisuus oli yhteydessä parempaan kognitioon pojilla.
Tutkimus toi uutta ja ennen julkaisematonta tietoa fysiologisen adrenarken yleisyydestä, ilmiasusta ja vaikutuksista normaaliväestössä.
Luokitus: WS 450, WS 440, WK 755, QU 107
Yleinen suomalainen ontologia: adrenarke; androgeenit; lapsen kehitys; sydän- ja verisuonitaudit; riskitekijät; kasvutekijät: sukupuolierot; kognitio; liikunta; ravitsemus
In Memory of Virpi
ACKNOWLEDGEMENTS
I conducted the research for this dissertation at the Department of Paediatrics, Kuopio University Hospital and the Institute of Clinical Medicine, Faculty of Health Science, the University of Eastern Finland from 2011 to 2020. This study was a part of the PANIC study from the Institute of Biomedicine, the University of Eastern Finland. I received financial support from the Finnish Cultural Foundation, the Foundation for Paediatric Research, Kuopio University Hospital, the Finnish Medical Foundation, the Päivikki and Sakari Sohlberg Foundation and the Rauha and Jalmari Ahokas Foundation.
I wish to thank all my supervisors from my heart. First of all, I want to thank my main supervisor Professor Jarmo Jääskeläinen. He guided me through this project during these years with his intelligent spirit and straightforward attitude. Likewise, I want to thank Professor Timo Lakka, the head of the PANIC study, for his innovative thoughts and supporting words in all phases of this study. My third supervisor Docent Virpi Lindi passed away one year before my dissertation. I could have never been able to complete this work without her and her everlasting positive, caring and determined guidance. I dedicate this work to the memory of Virpi.
This thesis has been reviewed by Docents Marja Ojaniemi and Matti Hero. Their comments were invaluable and helped further improve this thesis.
I was lucky to be able to collaborate with the enthusiastic and talented PANIC study group. Their work was the foundation of my thesis. I thank my co-authors Docent Anna Viitasalo, Tuomo Tompuri, M.D, Docent Aino-Maija Eloranta, Docent Eero Haapala, Juuso Väistö, M.Sc, Docent Jyrki Ågren, Docent Sari Väisänen, Professor Tomi Laitinen, Merja Häkkinen, Ph.D, Professor Seppo Auriola, Søren Brage Ph.D and Professor Ken Ong. I especially thank data manager Panu Karjalainen, M.Sc for his help with data and Merja Atalay, secretary in the PANIC study group, for helping me in many ways during this work.
I sincerely thank Professor emeritus Raimo Voutilainen for his skilful advice when I wrote my first article. He is a trailblazer in Finnish adrenarche research. I express my gratitude to laboratory technician Leila Antikainen for her biochemical analyses of DHEAS.
I also sincerely thank Keith Hakso for the language revision of the previously unpublished part of this thesis.
I express my sincere thanks to Docent Pekka Riikonen, Head of the Department of Paediatrics, Kuopio University Hospital, for giving me the opportunity to carry out this study.
I am thankful to Saija Savininen, M.D and Tiina Hyvärinen, M.D. for substituting me in my daily work.
I am grateful for the encouragement from my colleagues and co-workers at the Department of Paediatrics and Forensic Psychiatric Unit for Children and
Varkaus. It was nice to share the working room in Canthia with research fellows. In particular, I thank my fellow researches Antti Saari, M.D, Ph.D., Marjo Karvonen, M.D., and Katri Backman, M.D., Ph.D., for their support and help during this study.
I thank all my friends for bringing joy to my life. I wish to thank ”Rose Team”
Hille, Kirsti, Outi, Tarja and Tiina for their long-lasting friendship since we were medical students. It is always inspiring to get together. I am thankful to Marja-Leena for her true friendship since childhood.
I am grateful to my late parents Orvokki and Matti for giving me some of their resilience and my brother Raimo just for being there. I am also grateful to my mother- in-law Kaija and sister-in-law Tarja for their love and support.
I wish to express my deepest thanks to my children Sakari, Aarni-Matti, Reeta- Mari, Pietari and Selma, who have been grown up during these years and my husband Pekka for his love, patience and encouragement.
Kuopio, September 2020 Aino Mäntyselkä
L I S T O F O R I G I N A L P U B L I C A T I O N S
This dissertation is based on the following original publications:
I Mäntyselkä A, Jääskeläinen J, Lindi V, Viitasalo A, Tompuri T, Voutilainen R, Lakka TA. The presentation of adrenarche is sexually dimorphic and modified by body adiposity. The Journal of Clinical Endocrinology & Metabolism 99:
3889-3894, 2014.
II Mäntyselkä A, Jääskeläinen J, Eloranta A-M, Väistö J, Voutilainen R, Ong K, Brage S, Lakka TA, Lindi V. Associations of lifestyle factors with serum dehydroepiandrosterone sulphate and insulin-like growth factor-1 concentration in prepubertal children. Clinical Endocrinology 88: 234-242, 2018.
III Mäntyselkä A, Lindi V, Viitasalo A, Eloranta A-M, Ågren J, Väisänen S, Voutilainen R, Laitinen T, Lakka TA*, Jääskeläinen J*. Associations of dehydroepiandrosterone sulfate with cardiometabolic risk factors in
prepubertal children. The Journal of Clinical Endocrinology & Metabolism 103:
2592-2600, 2018.
IV Mäntyselkä A, Haapala EA, Lindi V, Häkkinen MR, Auriola S, Jääskeläinen J, Lakka TA. Associations of IGF-1 and adrenal androgens with cognition in childhood. Hormone Research in Paediatrics: 91:329-335, 2019.
The publications were adapted with the permission of the copyright owners.
* shared last authorship
C O N T E N T S
ABSTRACT ... 7
TIIVISTELMÄ ... 9
ACKNOWLEDGEMENTS ...13
1 INTRODUCTION ...21
2 REVIEW OF THE LITERATURE ...23
2 . 1 A d r e n a l d e v e l o p m e n t , s t r u c t u r e a n d f u n c t i o n . . . 2 3 2 . 1 . 1 A d r e n a l d e v e l o p m e n t . . . 2 3 2 . 1 . 2 A d r e n a l s t r u c t u r e . . . 2 4 2 . 1 . 3 A d r e n a l s t e r o i d o g e n e s i s .. . . 2 6 2 . 1 . 4 S t e r o i d o g e n e s i s i n a d r e n a r c h e . . . 2 8 2 . 1 . 5 P e r i p h e r a l m e t a b o l i s m a n d a c t i o n o f a d r e n a l a n d r o g e n s . . . 3 1 2 . 1 . 6 R e g u l a t i o n o f a d r e n a r c h e .. . . 3 3 2 . 2 P r e m a t u r e a d r e n a r c h e . . . 3 4 2 . 3 D H E A S a n d I G F - 1 . . . 3 5 2 . 3 . 1 D H E A S . . . 3 5 2 . 3 . 2 I G F - 1 . . . 3 6 2 . 4 A d i p o s i t y a n d l i f e s t y l e f a c t o r s a s s o c i a t e d w i t h a d r e n a r c h e .. . . 3 7 2 . 4 . 1 A d i p o s i t y .. . . 3 7 2 . 4 . 2 D i e t a r y f a c t o r s . . . 3 9 2 . 4 . 3 P h y s i c a l a c t i v i t y a n d s e d e n t a r y b e h a v i o r . . . 3 9 2 . 5 A s s o c i a t i o n s o f a d r e n a r c h e w i t h c h i l d h o o d h e a l t h . . . 4 0 2 . 5 . 1 E f f e c t s o n g r o w t h , p u b e r t a l t i m i n g a n d b o d y c o m p o s i t i o n . . . 4 0 2 . 5 . 2 A s s o c i a t i o n s w i t h c a r d i o m e t a b o l i c r i s k f a c t o r s .. . . 4 2 2 . 5 . 3 A s s o c i a t i o n s D H E A / D H E A S a n d I G F - 1 w i t h c o g n i t i o n a n d q u a l i t y o f l i f e .. . . 4 6 2 . 6 L o n g - t e r m e f f e c t s o f p r e m a t u r e a d r e n a r c h e .. . . 4 7 3 AIMS OF THE STUDY ...49
4 THE PRESENTATION OF ADRENARCHE IS SEXUALLY DIMORPHIC AND MODIFIED BY BODY ADIPOSITY ...51
4 . 1 A b s t r a c t . . . 5 1 4 . 2 I n t r o d u c t i o n .. . . 5 2 4 . 3 S u b j e c t s a n d M e t h o d s .. . . 5 3 4 . 4 R e s u l t s .. . . 5 4 4 . 5 D i s c u s s i o n . . . 5 9 4 . 6 A c k n o w l e d g e m e n t s . . . 6 0 5 ASSOCIATIONS OF LIFESTYLE FACTORS WITH SERUM DEHYDROEPIANDROSTERONE SULFATE AND INSULIN-LIKE GROWTH FACTOR-1 CONCENTRATION IN PREPUBERTAL CHILDREN ...61 5 . 1 S u m m a r y . . . 6 1
5 . 2 I n t r o d u c t i o n .. . . 6 2 5 . 3 S u b j e c t s a n d M e t h o d s .. . . 6 3 5 . 4 R e s u l t s .. . . 6 6 5 . 5 D i s c u s s i o n . . . 7 8 5 . 6 A c k n o w l e d g e m e n t s . . . 8 0 6 ASSOCIATIONS OF DEHYDROEPIANDROSTERONE SULFATE WITH
CARDIOMETABOLIC RISK FACTORS IN PREPUBERTAL CHILDREN ...83 6 . 1 A b s t r a c t . . . 8 3 6 . 2 I n t r o d u c t i o n .. . . 8 4 6 . 3 S u b j e c t s a n d M e t h o d s .. . . 8 5 6 . 4 R e s u l t s .. . . 8 7 6 . 5 D i s c u s s i o n . . . 9 4 6 . 6 A c k n o w l e d g m e n t s . . . 9 7 7 ASSOCIATIONS OF IGF-1 AND ADRENAL ANDROGENS WITH COGNITION
IN CHILDHOOD ...99 7 . 1 A b s t r a c t . . . 9 9 7 . 2 I n t r o d u c t i o n .. . . 1 0 0 7 . 3 M a t e r i a l s a n d M e t h o d s . . . 1 0 1 7 . 4 R e s u l t s .. . . 1 0 3 7 . 5 D i s c u s s i o n a n d c o n c l u s i o n . . . 1 0 7 7 . 6 A c k n o w l e d g m e n t . . . 1 0 9 8 GENERAL DISCUSSION ...111
8 . 1 S u m m a r y . . . 1 1 1 8 . 1 . 1 S t u d y s e t t i n g a n d p o p u l a t i o n .. . . 1 1 1 8 . 1 . 2 S e x u a l d i m o r p h i s m o f a d r e n a r c h e . . . 1 1 1 8 . 1 . 3 A s s o c i a t i o n s o f l i f e s t y l e f a c t o r s w i t h a d r e n a r c h e . . . 1 1 2 8 . 1 . 4 D H E A S a n d c a r d i o m e t a b o l i c r i s k . . . 1 1 3 8 . 1 . 5 A s s o c i a t i o n s o f a d r e n a l m a t u r a t i o n a n d I G F - 1 w i t h c o g n i t i o n .. . . 1 1 4 8 . 2 S t r e n g h t s a n d l i m i t a t i o n s o f t h e p r e s e n t s t u d y . . . 1 1 5 8 . 3 F u t u r e p e r s p e c t i v e s .. . . 1 1 5 9 CONCLUSIONS ...117 REFERENCES ...118
A B B R E V I A T I O N S
A4 Androstenedione ACAT Acyl-coenzyme A:
cholesterol acyltransferase ACTH Adrenocorticotropic
hormone
ALT Alanine aminotransferase AR Androgen receptor BMI Body mass index DXA Dual-energy X-ray
absorptiometry
DHEA Dehydroepiandrosterone DHEAS Dehydroepiandrosterone
sulfate
DHT Dihydrotestosterone HDL High-density lipoprotein HOMA-IR Homeostatic Model
Assessment for Insulin Resistance
hsCRP High-sensitivity C-reactive protein
HSL Hormone-sensitive lipase HSD Hydroxysteroid
dehydrogenase enzyme IGF-1 Insulin-like growth factor 1 11OHT 11β-hydroxytestosterone
IGFBP Insulin-like growth factor- binding protein
IQ Intelligence quotient LDL Low-density lipoprotein LC-MS/MS Liquid chromatography - tandem mass spectrometry PA Premature adrenarche P450scc Cytochrome cholesterol side
chain cleavage enzyme P450c17 Cytochrome P450c17 enzyme
PCOS Polycystic ovary syndrome POR Cytochrome P450
oxidoreductase PP Premature pubarche SGA Small for gestational age STAR Steroidogenic acute
regulatory protein SULT2A1 Sulfotransferase TC Total cholesterol TG Plasma triglycerides
VLDL-C Very low-density lipoprotein cholesterol
1 INTRODUCTION
Adrenarche is a gradually developing process of the adrenal cortex in which the zona reticularis starts to produce increasing amounts of androgen precursors, mainly dehydroepiandrosterone (DHEA), its sulfate (DHEAS), and androstenedione (A4) (Idkowiak et al. 2011, Voutilainen and Jääskeläinen 2015, Oberfield et al. 2018). These androgen precursors are converted peripherally to more potent androgens, testosterone and dihydrotestosterone (DHT), which are able to activate the androgen receptor (AR). Adrenarche occurs only in humans and some other higher primates, such as chimpanzees. The onset of adrenarche is gradual, starting as early as the first five years of life (Remer et al. 2005). The clinical signs of adrenarche include adult- type body odor, oily hair, acne, comedones, as well as pubic and axillary hair. If the clinical signs of adrenarche appear before the age of eight years in a girl or nine years in a boy (Silverman et al. 1952, Voutilainen et al., Ibáñez et al. 2000b) and serum DHEAS concentration is ≥ 1µmol/L (Utrtiainen et al. 2009) or 40 µg/dL ( Rosenfield 2007), a child is considered to have premature adrenarche (PA). In isolated biochemical adrenarche, serum DHEAS is above the reference range without clinical signs of adrenarche.
PA and an elevated serum DHEAS concentration have been associated with low birth weight, enhanced early growth (Tenhola et al. 2002, Ong et al. 2004, Neville and Walker 2005, Utriainen et al. 2009b), and increased adiposity in childhood and adolescence (Shi et al. 2009, Corvalán et al. 2013). Mild or moderate insulin resistance is a common finding in PA (Oppenheimer et al. 1995, Denburg et al. 2002), and some children with PA may be at an increased risk for metabolic syndrome, type 2 diabetes, and cardiovascular disease in adulthood (Utriainen et al. 2007, Mathew et al. 2008, Ibáñez et al. 2009).
The incidence of PA is markedly higher in girls than in boys with the ratio being up to 10:1 (Williams et al. 2011, Leung and Robson 2008), but until now no explanation for this unequal sex ratio has been given. Little is also known about the prevalence of PA in general populations of children. In a cross-sectional study among Lithuanian school girls, PA was uncommon, the prevalence being 0.8% at the age of 7-7.9 years (Žukauskaite et al. 2005). The prevalence of pubarche before the age of 8 years was 2.8% in white American girls (Herman-Giddens et al. 1997). The variation in the prevalence of precocious pubarche seems to be dependent on the children’s ethnic background. In an American study, only 0.01% of non-Hispanic white girls had pubic hair (Tanner stage ≥3) by the age of 8 years, but 3.0% of black girls and 1.3% of Mexican-American girls of the same age had it (Rosenfield et al. 2009).
However, these studies on pubarche did not report the prevalence of milder signs of androgen action.
Adrenarche occurs in all healthy children, but its regulation is not well understood. Most previous studies have focused on PA or premature pubarche (PP),
children. The purpose of this doctoral thesis was to study adrenarche in a general population of prepubertal Finnish girls and boys as part of the Physical Activity and Nutrition in Children (PANIC) study. The specific aims were to study the presentation of adrearche in girls and in boys, the associations of serum DHEAS with adiposity and other cardiometabolic risk factors, the associations of lifestyle factors, such as diet, physical activity and sedentary behavior with serum DHEAS and IGF- 1, and the associations of serum DHEAS and IGF-1 with cognition.
2 REVIEW OF THE LITERATURE
2.1 ADRENAL DEVELOPMENT, STRUCTURE AND FUNCTION
2.1.1 Adrenal development
Human adrenal development is a continuum beginning around the fourth week of gestation and continuing into adult life (Mesiano and Jaffe 1997). The adrenal gland develops originally from two separate embryological tissues: the medulla is derived from neural crest cells, whereas the cortex develops from the intermediate mesoderm, deriving from the urogenital ridge and having a common embryologic origin with the gonads and the kidney (Xing et al. 2015, Stewart and Newell-Price 2016).
Figure 1 elucidates the development of the adrenal gland.
F i g u r e 1 . S c h e m a t i c d i a g r a m o f t h e h u m a n a d r e n a l c o r t e x d u r i n g p r e n a t a l a n d p o s t n a t a l l i f e . M o d i f i e d f r o m S t e w a r t a n d N e w e l l - P r i c e ( 2 0 1 6 ) . D Z , d e f i n i t i v e z o n e ; F Z , f e t a l z o n e ; Z G , z o n a g l o m e r u l o s a ; Z R , z o n a r e t i c u l a r i s .
There are five landmark phases in the development of the adrenal cortex into different zones (Sucheston and Cannon 1968). The first three phases appear during the first trimester of pregnancy. The condensation of the coelomic epithelium occurs already within 3-4 weeks of gestation, whereas the proliferation and migration of coelomic epithelial cells occur at 4-6 weeks of gestation. The adrenogonadal primordium can been seen as the medial part of the urogenital ridge already at 4 weeks of gestation (Stewart and Newell-Price 2016). The adrenal and gonadal cells then separate (Miller and Flück 2014). At 8-10 weeks of gestation, fetal adrenal cortical cells differentiate into two distinct zones called the definitive zone and the fetal zone. The transitional zone is a narrow third layer between the definitive zone and fetal zone. The enlargement of the fetal zone accounts for the majority of the
adrenal prenatal growth, especially during the last six weeks of pregnancy, when the fetal adrenal gland is nearly the size of a kidney (Xing et al. 2015). In a fetus, the growth and function of the human adrenal cortex are regulated by Adrenocorticotropic hormone (ACTH), which is secreted from the fetal pituitary gland (Mesiano and Jaffe 1997). The fetal zone and the transitional zone synthesize androgen precursors, especially DHEAS, which serve as estrogen precursors in the placenta (Voutilainen and Jääskeläinen 2015). From mid-gestation the fetal adrenal gland expresses 3β-hydroxysteroid dehydrogenase (3-HSD) enzyme, which enables the fetal zone and the transitional zone to produce glucocorticoids, whereas the definitive zone produces mineralocorticoids (Serón-Ferré and Jaffe 1981, Mesiano and Jaffe 1997).
The fourth phase in the development of the adrenal cortex is the decline and disappearance of the fetal zone during the first months after birth. The definitive zone creates the zona fasciculata and the zona glomerulosa (Serón-Ferré and Jaffe 1981, Mesiano and Jaffe 1997). The innermost layer of the adrenal cortex, the zona reticularis, starts to develop more slowly from small local islets at the age of 2-3 years (Dhom 1973, Stewart and Newell-Price 2016).
The fifth phase of the development of the adrenal cortex is the establishment and stabilization of the adult zones (zona glomerulosa, zona fasciculata, zona reticularis) at the age of 10-20 years. The results of a large autopsy study demonstrated that proliferative adrenocortical cells were detected in the zona glomerulosa and the zona fasciculata before the age of 4 years and in the zona reticularis after the age of 4 years, but the number of these cells markedly decreased at the age of around 20 years (Hui et al. 2009). The development of human adrenal gland is thus characterized by rapid growth, high steroidogenic activity, and unique morphology (Mesiano and Jaffe 1997).
2.1.2 Adrenal structure
The adult adrenal gland has a pyramidal structure lying immediately above the kidney. The adrenal cortex consists of three histologically recognizable zones, zona glomerulosa, the zona fasciculata, and the zona reticularis. The zona glomerulosa lies immediately below the capsule, the zona fasciculata is in the middle, and the zona reticularis lies next to the medulla (Figure 2) (Stewart and Newell-Price 2016).
F i g u r e 2 . S c h e m a t i c d i a g r a m o f t h e s t r u c t u r e o f t h e h u m a n a d r e n a l c o r t e x . M o d i f i e d f r o m S t e w a r t a n d N e w e l l - P r i c e ( 2 0 1 6 ) .
The zona glomerulosa constitutes approximately 15% and the zona fasciculata approximately 75% of the cortex in older children and adults (Miller and Flück 2014).
Cells in the zona glomerulosa are smaller and more clustered than in the other zones (Stewart and Newell-Price 2016). Cells in the zona fasciculata are bigger and cells in the zona reticularis are arranged as cords, resulting a net-like shape that gives the zone its name reticulum (Xing et al. 2015). Arterial blood is provided from small
arteries arising from the renal and phrenic arteries and the aorta, entering the sinusoidal circulation of the cortex, and draining towards the medulla. The left adrenal vein drains to the left renal vein and the right adrenal vein drains directly to the vena cava (Figure 3) (Kahn and Angle 2010, Miller and Flück 2014).
F i g u r e 3 . A d r e n a l g l a n d a n a t o m y m o d i f i e d f r o m K a h n a n d A n g l e ( 2 0 1 0 ) .
2.1.3 Adrenal steroidogenesis
The outer layer of the adrenal cortex, the zona glomerulosa, produces mineralocorticoids (aldosterone, deoxycorticosterone), the zona fasciculata produces glucocorticoids (cortisol, corticosterone) and the innermost layer, the zona reticularis, produces androgen precursors (DHEA, DHEAS, A4) (Miller 2009, Miller and Flück 2014). The adrenal medulla secretes epinephrine (adrenaline), norepinephrine (noradrenaline), and a small amount of dopamine in response to stimulation by sympathetic preganglionic neurons. A few months after birth, apoptosis of the fetal zone causes a rapid fall in circulating androgens, but the zona glomerulosa and the zona fasciculata continue secreting mineralocorticoids and glucocorticoids.
Adrenal steroidogenesis is a dynamic process, in which cholesterol, the precursor for all steroids, is efficiently converted along a series of steps to the final product (Turcu and Auchus 2015). The proteins required for human adrenal steroidogenesis are steroidogenic acute regulatory protein (StAR), cytochrome P450
cholesterol side chain cleavage enzyme (cytochrome P450scc, CYP11A1) and other distinct cytochrome P450 enzymes such as cytochrome P450c11 isoenzymes P450c11β and P450c11AS (CYP11B1, 11β-hydroxylase), cytochrome P450c17 (CYP17A1, 17α-hydroxylase/17,20-lyase) and cytochrome P450c21 (CYP21A, 21- hydroxylase), 3-hydroxysteroid dehydrogenase (3-HSD), cytochrome P450 oxidoreductase (POR), cytochrome b5, sulfotransferase (SULT2A1), and steroid sulfatase (STS) (Miller 2009, Miller and Auchus 2011, Miller and Flück 2014).
Cholesterol derived from one’s diet and used in steroidogenesis is mainly transported in plasma low-density lipoproteins (LDL) (Gwynne et al. 1982), although steroidogenic cells can also synthesize cholesterol in the endoplasmic reticulum (Miller and Bose 2011). Figure 4 elucidates the cholesterol supply for use in steroidogenesis.
F i g u r e 4 . C h o l e s t e r o l s u p p l y t o m i t o c h o n d r i a f o r u s e i n s t e r o i d o g e n e s i s , m o d i f i e d f r o m M i l l e r a n d B o s e ( 2 0 1 1 ) . O M M , o u t e r m i t o c h o n d r i a l m e m b r a n e ; I M M , i n n e r m i t o c h o n d r i a l m e m b r a n e ; L D L , l o w - d e n s i t y l i p o p r o t e i n ; L A L , l y s o s o m a l a c i d l i p a s e ; L D L , l o w - d e n s i t y l i p o p r o t e i n ; A C A T , A :c h o l e s t e r o l a c y l t r a n s f e r a s e ; H S L , h o r m o n e - s e n s i t i v e l i p a s e ; S t A R , s t e r o i d o g e n i c a c u t e r e g u l a t o r y p r o t e i n .
The protein portion of LDL is hydrolyzed to free amino acids, and cholesterol esters are hydrolyzed to free cholesterol by lysosomal acid lipase (Miller and Bose 2011). Unesterified cholesterol can then be used as a substrate for steroidogenesis.
Cholesterol can be reesterified by acyl-coenzyme A:cholesterol acyltransferase (ACAT) and stored in lipid droplets and then released from lipid droplets by hormone-sensitive lipase (HSL). ACTH stimulates HSL and inhibits ACAT, thus increasing the availability of free cholesterol for steroid hormone synthesis (Miller and Auchus 2011). StAR transports cholesterol from the outer to the inner mitochondrial membrane, where cytochrome P450scc enzyme converts insoluble cholesterol to soluble pregnenolone (Miller and Bose 2011, Miller and Auchus 2011,
Miller 2017). Pregnenolone can be futher converted to other steroids depending on the enzyme profile of the adrenocortical cell (Figure 5) (Miller and Flück 2014, Miller 2009). Cytochrome P450c11 isoenzymes P450c11β and P450c11AS, also found in the mitochondria, catalyze 11β-hydroxylase, 18-hydroxylase and 18-methyloxidase activities. Cytochrome P450c17, found in the endoplasmic reticulum, has both 17 α- hydroxylase and 17,20-lyase activities. P450c21 catalyzes the 21-hydroxylation of progesterone and 17α-hydroxyprogesterone. These enzymes are involved in the formation of aldosterone from 11-deoxycorticosterone in the zona glomerulosa, and cortisol from 11-deoxycortisol in the zona glomerulosa (Miller 2009, Miller and Flück 2014). Each of the P450 reactions are catalyzed by HSD, including two isoenzymes, 3α- and 3β-hydroxysteroid dehydrogenases. Cytochrome P450c17 requires POR as an electron donor, and adrenodoxin reductase and adrenodoxin also serve as electron transfer proteins for P450s (Miller 2009).
2.1.4 Steroidogenesis in adrenarche
Adrenarche refers to a maturational increase in the secretion of adrenal androgen precursors, mainly DHEA, DHEAS and A4. The onset of adrenarche is gradual, and it begins often during the first five years of life (Palmert et al. 2001, Remer et al. 2005), although in the past it has been proposed to occur at around 6-8 years of age, without marked changes in ACTH and cortisol secretion (Dhom 1973, Reiter et al. 1977, Miller and Auchus 2011, Guran et al. 2015). The increased secretion of adrenal adrogens is associated with morphological changes in the zona reticularis. These changes include the increased expression of cytochrome b5 and SULT2A1 and the decreased expression of 3β-HSD2 (Suzuki et al. 2000, Hui et al. 2009).
The first step in steroidogenesis related to adrenarche is the conversion of cholesterol to pregnenolone. This involves three distinct chemical reactions: 20α- hydroxylation, 22-hydroxylation, and the scission of the cholesterol side-chains (Miller 2009). Then pregnenolone undergoes 17α-hydroxylation to yield 17- hydroxypregnelone or 17-hydroxyprogesterone. The 17,20-lyase activity of cytochrome P450c17 prefers 17–hydroxypregnenolone to produce DHEA (Miller and Auchus 2011). The 17,20-lyase activity of cytochrome P450c17 is regulated by cytochrome b5 and mitogen-activated protein kinase 14 (Tee et al. 2013). SULT2A1 catalyzes the sulfation of DHEA. DHEAS is more stable than DHEA in systemic circulation and has a 100-1,000-fold higher serum concentration than DHEA (Miller 2017). Figure 5 elucidates pathways of adrenocortical steroid synthesis.
igure 5. Steroid synthesis pathway in the adrenal cortex and peripheral steroid metabolism (modified from Miller 2009).
Only a small amount of 17-hydroxyprogesterone is converted to A4 catalyzed by cytocrome P450c17 (Miller 2009). The secretion of DHEA and DHEAS increases during and after puberty and reaches its peak in young adulthood (Orentreich et al.
1984). Figure 6 elucidates the variation on the circulating DHEAS concentration throughout human life.
F i g u r e 6 . C o n c e n t r a t i o n s o f D H E A S a s a f u n c t i o n o f a g e . 3 7 µ g / d L = 1 µ m o l / L . M o d i f i e d f r o m M i l l e r a n d F l ü c k ( 2 0 1 6 ) .
Taken together, cytochrome P450c17, which possesses both 17α-hydroxylase and 17,20- lyase activities, is the key point in adrenal androgenic steroidogenesis. No cytochrome P450c17 activity is present in the zona glomerulosa, whereas 17α- hydroxylase but not 17,20-lyase activity is present in the zona fasciculata. Both cytochrome P450c17 activities are present in the zona reticularis and pregnenolone is converted to sex steroids, mainly to DHEAS and DHEA (Miller and Auchus 2011).
Recently, liquid chromatograph - tandem mass spectrometry (LC-MS/MS) has revealed the role of more androgenic steroids than traditional DHEA, DHEAS and A4 in adrenarche. These include 11-oxygenated androgens, 11β- hydroxyandrostenedione (11OHA4), 11-ketoandrostenedione (11KA4), 11β- hydroxytestosterone (11OHT), and 11-ketotestosterone (11KT) (Rege et al. 2018).
11OHA4 is the second most abundant unconjugated androgen after DHEA derived from the adrenal gland. Its metabolite 11-ketotestosterone is mostly produced in peripheral tissues (Turcu et al 2020). 11-ketosterone and its 5α-reduced product 11- ketodihydrotestosterone have bioactivity that is similar to that of testosterone and dihydrotestosterone (Turcu et al. 2020). Circulating concentrations of 11KA4 and 11KT increase during adrenarche (Rege et al. 2018). Oxygenated androgens seem to be important also in some other well-known hyperandrogenic disorders such as
congenital adrenal hyperplasia and polycystic ovary syndrome (PCOS) (Turcu et al 2020). Serum testosterone levels do not rise markedly in adrenarche (Kulle et al.
2010).
2.1.5 Peripheral metabolism and action of adrenal androgens
DHEA, DHEAS, and A4 are androgen precursors and activate the adrenal receptor (AR) only weakly (Chen et al. 2004 and 2005). They are converted to more potent androgens, testosterone and dihydrotestosterone (DHT), in the peripheral tissues to cause the clinical signs of adrenarche: adult type body odor, oily hair, acne, comedones, as well as axillary and pubic hair (Rosenfield and Lucky 1993, Utriainen et al. 2009a). The discovery of this peripheral synthesis led to the coining of the term intracrinology (Labrie et al. 1995 and 2001). The AR is an X-chromosome-encoded, ligand-activated intracellular transcription factor that belongs to the steroid/nuclear reseptor superfamily (Zouboulis et al. 2007, McEwan and Brinkman 2000). The AR is expressed widely in the skin, including epidermal and follicular keratinocytes, sweat gland cells, sebocytes, dermal fibroblasts, dermal papilla cells, endothelial cells, and genital melanocytes (Zouboulis et al. 2007). Testosterone and DHT stimulate the development of apocrine glands to produce adult type body odor and oily hair and the secretion of the sebaceous glands, leading to acne and comedones (Rosenfield and Lucky 1993 , Zouboulis et al. 2007, Tóth et al. 2011). The growth of axillary and pubic hair (pubarche) is the classical phenotypic hallmark of adrenarche (Rosenfield and Lucky 1993, Auchus and Rainey 2004, Rege and Rainey 2012). Figure 7 elucidates the conversion of DHEA, DHEAS, and A4 to more potent androgens, testosterone and DHT, in the peripheral tissues in peripheral cells.
F i g u r e 7 . P e r i p h e r a l m e t a b o l i s m a n d t h e a c t i o n o f a d r e n a l a n d r o g e n s ( m o d i f i e d f r o m U t r i a i n e n e t a l . 2 0 1 5 ) . 1 ) A n a l t e r n a t i v e b a c k d o o r p a t h w a y i n D H T f o r m a t i o n ( K a m r a t h e t a l . 2 0 1 2 ) . 2 ) A n alternative pathway from A4 to bioactive androgen 5α,11β- h y d r o x y t e s t o s t e r o n e ( 5 , 1 1 O H T ) v i a 11β- h y d r o x y a n d r o s t e n e d i o n e ( 1 1 O H A 4 ) ( R e g e a n d R a i n e y 2 0 1 2 ) . 3 ) T h e a c t i v i t y o f A R i s i n f l u e n c e d b y AR g e n e p o l y m o r p h i s m s , e p i g e n e t i c m o d u l a t i o n s , a n d t h e e x p r e s s i o n o f i t s c o f a c t o r s ( V o t t e r o e t a l . 2 0 0 6 , L a p p a l a i n e n e t a l . 2 0 0 8 a ) .
In peripheral tissues, steroid sulfatase hydrolyzes DHEAS to DHEA as the first step in the conversion of DHEAS to testosterone and DHT (Miller 2009, Miller and Auchus 2011). 3β-HSD converts DHEA to A4 and then A4 is converted to testosterone by 17β-hydroxysteroid dehydrogenase (17β-HSD). 5α-reductase converts testosterone to DHT. Furthermore, A4 and testosterone can be converted to estrogens (estrone and estradiol) by aromatase enzyme (P450aro, CYP19A1).
Studies on the mechanisms of virilization have also revealed the “backdoor pathway” that leads 17OHP to DHT without going through A4 or testosterone (Auchus 2004, Miller and Auchus 2011, Kamrath et al. 2012, Miller and Auchus 2019).
This pathway will function only when both 5α-reductase and cytochrome P450c17 are expressed in the same tissue (Auchus 2004).
The clinical signs of adrenarche typically appear when DHEAS and other adrenal androgens reach a certain activity level. However, the appearance of these signs may
vary depending on the peripheral conversion and sensitivity of these androgens.
There are ethnic differences in adrenal androgen production (Girgis et al. 2000). Most reports on the clinical signs of adrenarche have focused on pubarche, although other signs of adrenarche are more common. Tanner and co-workers found in the 1960’s that Caucasian girls reached Tanner stage 2 in pubic hair development (P2) at the age of 11.7 years on average (Marshall and Tanner 1969). The mean age for pubic or axillary hair appearance was 11.0 years in Lithuanian schoolgirls (Žukauskaite et al.
2005). Guran and coworkers found in a Turkish pediatric population that biochemical adrenarche (serum DHEAS level over 40 µg/dL) started one year earlier in boys, but higher DHEAS levels were needed for transition from P1 to P2 in boys than in girls (Guran et al. 2015). In this Turkish cohort, median serum DHEAS levels in P2 were 63 µg /dL in girls and 90 µg /dL in boys. Similarly, in an American study among girls reaching pubarche at the mean age of 9.7 years, the mean serum concentration of DHEAS was 97 µg /dL (2.6 µmol/L) ( Biro et al. 2008). In a Finnish study among children with PA, serum androgen concentrations were highest in children with pubic and axillary hair (median DHEAS 2.2 µmol/L or 81 µg /dL) (Utriainen et al. 2009a). However, they were also increased in children with milder signs of adrenarche (median DHEAS 1.7 µmol/L or 63.0 µg /dL) when compared to the control group (0.7 µmol/L or 25.9 µg /dL). In this study, some children had milder signs of androgen action with relatively low androgen concentrations. In a recent Portuguese cohort of 100 children with PA, 94% of whom had pubarche, serum DHEAS levels were similar in 85 girls and in 15 boys. However, pubic hair appeared the girls at a median age of 6 years and in the boys at a median age of 7 years (Santos et al. 2019).
Children may also have increased serum DHEAS concentrations without clinical signs of adrenarche (Rosenfield 1994, Vottero et al. 2006 , Utriainen et al. 2009a).
Measured circulatory androgen bioactivity in children with PA was low without differing significantly from the control group in a recent Finnish study, supporting the hypothesis that the peripheral conversion of androgens is indeed essential for clinical androgenic effects (Liimatta et al. 2014) and that the clinical signs of adrenarche do not fully reflect serum DHEAS concentrations.
2.1.6 Regulation of adrenarche
The ultimate trigger for adrenarche remains unknown. ACTH regulates adrenal steroidogenesis by responding to the cholesterol supply, and ACTH also has slowing effects on adrenal steroidogenesis by stimulating the expression of steroidogenic genes (Miller and Flück 2014). Despite huge increases in the secretion of DHEAS and DHEA, the circulating concentrations of ACTH do not change with age (Miller 2009).
Circulating ACTH levels were also similar between PA and control children in a Finnish study (Utriainen et al. 2009a). It has been suggested that intra-adrenal cortisol causes a marked stimulation of adrenal DHEA secretion by competively inhibiting 3β-HSD2 enzyme without a significant effect on the activity of 17,20-lyase enzyme (Majzoub and Tapor 2018). However, unchanged circulating cortisol levels have been
found during adrenarche (Parker et al. 1978, Kelnar and Brook 1983, Vandewalle et al. 2014).
Immunocytochemical studies have shown increasing expression of cytochrome b5 and SULT2A and decreasing expression of 3β-HSD2 during adrenarche (Miller 2009 and 2017), but the molecular trigger for adrenarche has not been identified. A variation in the gene encoding 3β-HSD1 was detected in different ethnic groups among adults (Wang et al. 2007). It has also been proposed that the serine/threonine phosphorylation of cytochrome P450c17 by cyclic AMP-dependent kinase accounts for the increase in 17,20-lyase activity that stimulates adrenal androgen secretion, and that this process could be initiated by increased IGF-1 or insulin activity (Zhang et al.
1995).
Factors regulating the peripheral conversion of androgens have been poorly studied. DHEAS is mostly protein-bound. An inverse correlation between sex hormone-binding globulin (SHBG) and androgen bioactivity was found in a Finnish study (Liimatta et al. 2014). SHBG binds estradiol, DHT, and especially testosterone, but not DHEAS, in the circulation and thereby partly determines sex steroid bioavailability in target tissues. Circulating SHBG levels are highest at the age of 5 and decline thereafter (Pinkney et al. 2014). In a Finnish study, PA children with clinical signs of adrenarche had shorter AR gene cytosine-adenine-guanine repeat lengths (CAGn) than healthy controls,leading to a higher AR activity (Lappalainen et al. 2008a)
The timing of adrenarche appears to be influenced by prenatal and postnatal factors such as fetal and childhood growth (Pere et al. 1995, Ong et al. 2004, Utriainen et al. 2009b). Nutritional status may also contribute to the development of adrenarche (Remer and Manz 1999).
2.2 PREMATURE ADRENARCHE
The definition for PA includes clinical signs of adrenarche appearing before eight years in a girl or nine years in a boy and serum DHEAS concentration ≥ 1µmol/L (Utriainen et al 2009a) or 40 µg/dL (Rosenfield 2007). Children with PA present with enhanced linear growth in childhood beginning as early as the age of 2 years (Pere et al. 1995). PA children are also often overweight (Utriainen et al. 2009b). Moreover, bone age is often advanced in PA children (Voutilainen et al. 1983, Sopher et al. 2011, Gurnurkar et al. 2014, Liimatta et al. 2017, Santos-Silva et al. 2019).
The prevalence of PA has been reported in a few studies, and it appears to vary in different populations (Banerjee et al. 1998, Rosenfield et al. 2009). In a Lithuanian study of school girls, PA was detected in only 0.8% of girls aged 7-7.9 years (Žukauskaite et al. 2005). The prevalence of pubarche before the age of 8 years was 2.8% in white American girls (Herman-Giddens et al. 1997.). The American study of Rosenfield and coworkers (2009) reported that only 0.01% of non-Hispanic white girls had pubic hair (Tanner stage ≥3) by the age of 8 years, but pubic hair was seen in 3.0% of black and 1.3% of Mexican-American girls. The reported prevalence of
premature pubarche in Turkey was 4.3% (Atay et al. 2012). The incidence of PA has been found to be markedly higher in girls than in boys with the ratio being up to 10:1 (Leung and Robson 2008, Williams et al. 2011).
Lower birth weight is associated with higher adrenal androgen concentrations in childhood (Ibáñez et al. 1999, Ghirri et al. 2001, Tenhola et al. 2002). The association of adiposity (Utriainen et al. 2007, Shi et al. 2009, Corvalán et al. 2013) and rapid early weight gain with PA has been shown in several studies (Ong et al. 2004, Mericq et al.
2017, Marakaki et al. 2017, Nordman et al. 2017, Na et al. 2018).
It has been shown that increased AR activity due to shorter AR gene cytosine- adenine-guanine repeat lengths (Vottero et al. 2006, Lappalainen et al. 2008a) and reduced AR gene methylation may explain the signs of androgen action in children with PA (Vottero et al. 2006). An ACTH receptor gene polymorphism has also been found to associate with the severity of premature androgen action (Lappalainen et al. 2008b).
PA has been associated with the components of metabolic syndrome (Oppenheimer et al. 1995, Ibáñez et al. 1998, Denburg et al. 2002, Potau et al. 2003, Utriainen et al. 2007, Mathew et al. 2008, Ibáñez et al. 2009) and functional ovarian hyperandrogenism (Rosenfield 2007, Ibáñez et al. 2009). Pubertal development, including menarche, has been observed to occur slightly earlier in children with PA than in children without it (Pere et al. 1995, Ibáñez et al. 2006, Remer et al. 2010, Liimatta et al. 2017). In a recent follow-up study among women, a history of PA without a distinct pubertal growth spurt was associated with normal adult height.
Moreover, body mass index (BMI) as well as serum DHEAS and IGF-1 concentrations were similar in women with a history of PA and in women without it at the mean age of 18 years (Liimatta et al. 2018b).
2.3 DHEAS AND IGF-1
2.3.1 DHEAS
DHEAS is the most abundant endogenous steroid hormone in systemic circulation and is a good marker of adrenal androgen secretion. Serum DHEAS level has no diurnal rhythm or day-to-day variation. The serum concentration of DHEAS is remarkably higher than the concentration of DHEA and many times higher than that of any other steroid hormone (Longcope 1996). Sulfation and desulfation influence the concentrations of DHEAS in various tissues. Sulfation pathways dominate in the adrenal gland, brain, colon, and the kidney (Mueller et al. 2015). The highest concentrations of sulfotransferases have been found in the liver and the intestine.
SULT2A1 is specifically expressed in the zona reticularis and is responsible for the large DHEAS production from the adrenal gland. The sulfonation by SULT2A1 requires a sulfate donor, 3´-phosphoadenosine-5´-phosphosulfate (Rege and Rainey 2012, Mueller et al. 2015). The molecular causes of androgen excess may rarely be due to a failure in the sulfation pathway that converts DHEA to DHEAS (Mueller et
al. 2015). The conversion of DHEA to DHEAS prevents the conversion of DHEA to more potent androgens.
The production of DHEAS regresses during the first postnatal months and is low in early childhood until adrenarche. In a study by Palmert and coworkers (2001), serum DHEAS levels increased by approximately 22% per year from the mean age of 2.9 years to the mean age of 12.3 years. Some studies have shown an earlier rise of serum DHEAS levels in girls than in boys (Ducharme et al.1976, Ilondo et al. 1982), whereas a Turkish study reported higher serum DHEAS levels in boys than in girls (Guran et al. 2015). However, no gender difference in serum DHEAS concentration was observed between prepubertal girls and boys in a Chilean study (Corvalán et al.
2013). Lower birth weight has been associated with higher adrenal androgen concentrations in childhood, and being born large for gestational age has been related to lower serum DHEAS concentrations in prepubertal children (Nordman et al. 2017).
In a recent Finnish study, children with the highest serum DHEAS levels at the age of one year had the highest DHEAS concentrations at the age of six years (Liimatta et al. 2020). Shorter birth length and more rapid catch-up growth in height during the first year of life were associated with higher serum DHEAS levels at the age of six years. DHEAS concentration declines with age, and at the age of 70 years circulating DHEAS concentrations have decreased by 90% compared to the peak levels achieved by the age of 20-30 years (Orentreich et al. 1984). A child is defined to have biochemical adrenarche if serum DHEAS is over 1µmol/L (Utriainen et al.
2009a) or 40 µg/dL (Rosenfield and Lucky 1993, Rosenfield 2007). Guran and coworkers suggested that the cut-off age for serum DHEAS levels rising above 40 µg/dL could be 8 years for girls but 7 years for boys (Guran et al. 2015). In this Turkish study among 500 healthy chidren, serum DHEAS levels remained at a low level between the ages of 6 months and 5 years and gradually increased thereafter during childhood. Likitmaskul and coworkers (1995) have used a term exaggerated adrenarche for PA children whose serum DHEAS concentrations are above 6 µmol/L that is the upper limit for the normal range (Likitmaskul et al. 1995).
Heritability of serum DHEAS is high; estimated heritability by model-fitting on data from 180-pairs is 0.61 (0.52-0.70) and in prepubertal children even higher, at 0.82 (0.71-0.90) (Li et al. 2016).
2.3.2 IGF-1
IGF-1 is a polypeptide hormone produced mainly by the liver. It has endocrine, paracrine, and autocrine effects (Puche and Castilla-Cortázar 2012). It mediates many actions of growth hormone that together with nutrition are the main factors controlling hepatic IGF-1 production. Serum IGF-1 levels in a newborn child are generally 30-50% of adult levels (Backeljauw et al. 2014). Serum IGF-1 concentration rises slowly during childhood and more so in puberty, reaching levels that are three times than in adults. Girls present peak IGF-1 values approximately one year earlier than boys (Löfqvist et al. 2005, Chaler et al. 2009).
IGF-1 binds to its type I receptor, which is a similar type of receptor as the insulin receptor. In addition to insulin, IGF-1 can also bind to the insulin receptor and thus have some insulin-like activity. IGF-1 circulates in plasma with a carrier protein insulin-like growth factor-binding protein. IGF-1 and its receptor are expressed in the adrenal cortex (Voutilainen and Miller 1987, Baquedano et al. 2005), and IGF-1 increases adrenal androgen production in cultured human adrenocortical cells (l'Allemand et al. 1996).
The involvement of IGF-1 in the production of adrenal androgens in children has been shown in girls with Turner syndrome, who presented increased serum levels of adrenal androgens in parallel with increased serum IGF-1 levels after beginning growth hormone therapy (Balducci et al. 1998). Children aged 7 years with higher serum DHEAS had higher serum IGF-1 than their counterparts of similar age in a longitudinal Chilean study among 972 children (Mericq et al. 2017). Children with PA have had increased serum IGF-1 concentrations in many studies (Ibáñez et al.
1998, Denburg et al. 2002, Silfen et al. 2002; Utriainen et al. 2009b). IGF-1 has been suggested to mediate the effect of excess weight on the initiation of adrenarche (Vuguin et al. 1999, l'Allemand et al. 2002; Guercio et al. 2003, Güven et al. 2005). On the other hand, elevated serum IGF-1 concentrations may also represent increased androgen action on a growth hormone - IGF-1 axis (Voutilainen and Jääskeläinen 2015). All in all, DHEAS, IGF-1, and growth velocity associate with each other, but the causality between these factors remains to be elucidated.
2.4 ADIPOSITY AND LIFESTYLE FACTORS ASSOCIATED WITH ADRENARCHE
2.4.1 Adiposity
The heritability of adrenal androgen secretion adjusted for body weight is high, but environmental factors also play a role in it (Pratt et al. 1994). A number of studies have shown a clear association between adiposity and adrenarche (Table 1).
T a b l e 1 . A d i p o s i t y a s s o c i a t e d w i t h a d r e n a r c h e .
Reference Subjects Ethnic
origin Findings
I b á ñ e z e t a l . 2 0 0 3
6 7 g i r l s w i t h a h i s t o r y o f
P P a n d 6 5 c o n t r o l s C a t a l a n P P g i r l s h a d a h i g h e r c e n t r a l f a t m a s s m e a s u r e d b y D X A
G a r n e t t e t a l . 2 0 0 4
2 5 5 h e a l t h y c h i l d r e n
a g e d 7 - 8 y e a r s A u s t r a l i a n
D H E A S a n d I G F - 1 c o r r e l a t e d p o s i t i v e l y w i t h b o d y f a t p e r c e n t a g e m e a s u r e d b y
D X A U t r i a i n e n e t a l .
2 0 0 7
7 3 P A c h i l d r e n a n d 9 8
c o n t r o l s F i n n i s h B M I S D S w a s h i g h e r i n P A c h i l d r e n
R o s e n f i e l d e t a l . 2 0 0 9
1 3 9 4 g i r l s a n d 1 3 1 3 b o y s a g e d 8 - 1 8 y e a r s
A m e r i c a n ( m i x e d )
H i g h e r B M I w a s a s s o c i a t e d w i t h e a r l i e r p u b a r c h e i n g i r l s
S h i e t a l . 2 0 0 9
1 3 7 h e a l t h y p r e p u b e r t a l c h i l d r e n a g e d 3 - 1 2
y e a r s
G e r m a n
E s t i m a t e d b o d y f a t p e r c e n t a g e w a s p o s i t i v e l y a s s o c i a t e d w i t h u r i n a r y a d r e n a l
a n d r o g e n s e c r e t i o n U t r i a i n e n e t a l .
2 0 0 9 c
6 4 P A c h i l d r e n a n d 6 2
h e a l t h y c o n t r o l s F i n n i s h
C h i l d r e n w i t h P A h a d a h i g h e r b o d y f a t p e r c e n t a g e m e a s u r e d b y B I A t h a n t h e i r
c o n t r o l s C o r v a l á n e t a l .
2 0 1 3
9 6 9 c h i l d r e n w i t h a
m e a n a g e o f 6 . 9 y e a r s C h i l e a n D H E A S c o r r e l a t e d p o s i t i v e l y w i t h b o d y f a t p e r c e n t a g e m e a s u r e d b y B I A A k y ü r e k e t a l .
2 0 1 5
1 0 0 o b e s e c h i l d r e n a n d 4 0 c o n t r o l s a g e d 6 - 1 8
y e a r s
T u r k i s h D H E A S c o r r e l a t e d p o s i t i v e l y w i t h w a i s t c i r c u m f e r e n c e a n d w a i s t t o h i p r a t i o C e b e c i a n d
Taș 2015
4 7 P A g i r l s a n d 5 7
c o n t r o l s T u r k i s h
P A g i r l s h a d h i g h e r w e i g h t a n d h i g h e r b o d y f a t p e r c e n t a g e m e a s u r e d b y B I A
t h a n t h e i r c o n t r o l s K i m e t a l . 2 0 1 6 2 4 2 g i r l s a g e d 7 - 1 3
y e a r s K o r e a n D H E A a n d A 4 c o r r e l a t e d p o s i t i v e l y w i t h
b o d y f a t p e r c e n t a g e m e a s u r e d b y B I A M e r i c q e t a l .
2 0 1 7
9 7 2 c h i l d r e n f o l l o w e d f r o m b i r t h t o 7 y e a r s o f
a g e
C h i l e a n H i g h e r D H E A S w a s a s s o c i a t e d w i t h h i g h e r B M I S D S
N a e t a l . 2 0 1 8 1 9 2 g i r l s a g e d 8 y e a r s K o r e a n D H E A S c o r r e l a t e d p o s i t i v e l y w i t h B M I S D S
C a o e t a l . 2 0 1 9
1 2 2 0 b o y s a g e d 6 - 1 0
y e a r s C h i n e s e D H E A , A 4 a n d t e s t o s t e r o n e w e r e h i g h e r
i n o v e r w e i g h t a n d o b e s e b o y s S a n t o s - S i l v a e t
a l . 2 0 1 9
1 0 0 P A c h i l d r e n w i t h a
m e a n a g e o f 7 . 4 y e a r s P o r t u g u e s e D H E A S a n d A 4 w e r e h i g h e r i n
o v e r w e i g h t a n d o b e s e P A c h i l d r e n D X A , d u a l - e n e r g y X - r a y a b s o r p t i o m e t r y ; B I A , b i o e l e c t r i c a l i m p e d a n c e .
2.4.2 Dietary factors
There are a few studies on the associations of dietary factors with adrenal androgen metabolism in children. In a German study, higher dietary animal protein content was associated with higher urinary excretion of adrenal C19 steroid metabolites (Shi et al. 2009).
There are more studies on the associations of dietary factors with IGF-1. A higher intake of protein (Rogers et al. 2005, Kerver et al. 2010), animal protein (Hoppe et al.
2004a; Rogers et al. 2005), and energy (Kerver et al. 2010) have been related to a higher serum IGF-1 concentration in healthy children. Also, a lower intake of mono- and polyunsaturated fat (Rogers et al. 2005) and a higher consumption of milk (Hoppe et al. 2004a and 2004b) have been associated with a higher serum IGF-1 concentration in children. There is also some evidence that the reduction of calories and protein in the diet decreases IGF-1 secretion, whereas in obesity, serum IGF-1 concentration is normal or elevated (Hawkes and Grimberg 2015).
2.4.3 Physical activity and sedentary behavior
There are no earlier studies on the association of physical activity or sedentary behavior with serum or urinary adrenal androgen levels in children or adolescents.
However, there are some plausible biological mechanisms for the associations of physical activity with adrenal androgens. Androgens are known to have direct anabolic effects on skeletal muscle (Enea et al. 2011). They increase muscle power, particularly when combined with exercise (Jardi et al. 2018). Androgens may also improve the adaptation of the body to exercise.
Some studies in adults have shown that exercise induces an increase in the circulating levels of androgens (Enea et al. 2011). Elevated serum DHEAS levels have also been found during such exercises as running marathons, triathlons, soccer, and swimming (Collomp et al. 2015). Enea and coworkers (2009) reported a marked rise in serum DHEAS levels in young women during a relatively short bout of intensive submaximal exercise. In premenopausal women and in men, acute exercise has been observed to increase circulationg levels of DHEAS and DHEA (Bonen and Keizer 1987, Kraemer et al. 2001, Tremblay et al. 2005). A resistance exercise session led to increased serum DHEA and DHEAS concentrations in women (Copeland et al. 2002, Aizawa et al. 2003). Exercise is a kind of a stress on the body, and the intensity and duration of this stress may differentially affect ACTH secretion (Enea et al. 2011).
However, there are contradictory results on whether regular exercise could modify serum DHEAS concentration at rest (Collomp et al. 2015).
There are some studies on the associations of physical activity with serum IGF-1 levels. There is some evidence that a single bout of exercise increases serum IGF-1 concentration (Berg and Bang 2004). Moreover, exercise training during positive energy balance elevated serum IGF-1 levels by increasing growth hormone secretion in children (Eliakim et al. 2001, Eliakim and Nemet 2013). However, exercise training during negative energy balance or heavy training decreased serum IGF-1
concentrations in children and adults (Berg and Bang 2004, Eliakim et al. 2001, Eliakim and Nemet 2013). Intensive training of young ice hockey players led to a marked increase in serum growth hormone concentration, whereas there was no change in serum IGF-1 levels (Kochanska-Dziurowicz et al. 2015).
To our knowledge, there are no previous studies on associations of sedentary behavior with serum DHEAS or IGF-1 concentration in children or adolescents.
2.5 ASSOCIATIONS OF ADRENARCHE WITH CHILDHOOD HEALTH
2.5.1 Effects on growth, pubertal timing and body composition
Androgens stimulate bone formation during growth, and ARs are widely expressed in bone tissue, most highly in osteoblasts and hypertophic chondrocytes of the growth plate (Abu et al. 1997). ARs were also observed in newly-formed osteocytes in the endothelial cells of blood vessels within the bone marrow. Both linear growth and the increase in bone mass are modified by androgens. Aromatization of androgens to estrogens mediates at least part of the anabolic effects on bones (Vanderschueren 1996). Adrenal androgen secretion, in its physiological range, has a role in the accretion of bone strength in healthy children even before pubarche (Remer et al. 2003). Serum levels of adrenal-derived steroids were positively associated with skeletal maturation indendently of age in a study by Vendewalle and coworkers (2014).
Most children with PA have a body height above average and have advanced bone age from 0.6 to over 2 years (Voutilainen et al. 1983, Ghizzoni and Milani 2000, Accetta et al. 2004, Charkaluk et al. 2004, Utriainen et al. 2009b, Paterson et al. 2010, von Oettingen et al. 2012, DeSalvo et al. 2013, Marakaki et al. 2017). Children with PA often use a greater part of their growth potential earlier than those with average timing of adrenarche (Pere et al. 1995). Utriainen and coworkers (2009b) reported a higher mean height already at the age of 1 year in girls with PA than age-matched control girls. PA children were taller than controls at prepuberty (Utriainen et al.
2009b) and at the age of 12 years (Liimatta et al. 2017), but not at 18 years (Liimatta et al. 2018b). The final height of PA children does not usually differ from mean parental height in spite of their reduced pubertal growth spurt (Voutilainen and Jääskeläinen 2015).
Rapid growth is a consequence of adrenarche, but childhood growth can also partly explain variations in adrenarche (Majzoub and Tabor 2018). Rapid catch-up growth was suggested as the reason for experiencing biochemical adrenarche two years earlier in first-generation Bangladeshi girls in the United Kingdom than in children who remained in Bangladesh (Houghton et al. 2014).
There is some evidence of an association between DHEAS and bone mineral density in children. Urinary C19 androgen metabolites were positively associated with cortical density, cortical area, and bone mineral content determined by peripheral quantitative computed tomography at the proximal radial diaphysis in a
