Recent Publications in this Series HANNA PAATELA Role of in High-Density Lipoprotein-Mediated Vasodilation and in Adipose Tissue Biosynthesis

7/2016 César Araujo Prostatic Acid as a Regulator of Endo/Exocytosis and Lysosomal Degradation 8/2016 Jens Verbeeren Regulation of the Minor Spliceosome through Alternative Splicing and Nuclear Retention of the U11/U12-65K mRNA 9/2016 Xiang Zhao dissertationes scholae doctoralis ad sanitatem investigandam HMGB1 (Amphoterin) and AMIGO1 in Brain Development universitatis helsinkiensis 26/2016 10/2016 Tarja Pääkkönen (Jokinen) Benign Familial Juvenile Epilepsy in Lagotto Romagnolo Dogs 11/2016 Nora Hiivala Patient Safety Incidents, Their Contributing and Mitigating Factors in Dentistry 12/2016 Juho Heinonen Intravenous Lipid Emulsion for Treatment of Local Anaesthetic and Tricyclic Antidepressant Toxicity 13/2016 Riikka Jokinen HANNA PAATELA Genetic Studies of Tissue-Specifc Mitochondrial DNA Segregation in Mammals 14/2016 Sanna Mäkelä Role of Dehydroepiandrosterone in Activation of Innate Immune Responses by Toll-like Receptors and Infuenza Viruses 15/2016 Mari Hirvinen High-Density Lipoprotein-Mediated Vasodilation Immunological Boosting and Personalization of Oncolytic Virotherapies for Cancer Treatment and in Adipose Tissue Steroid Biosynthesis 16/2016 Sofa Montalvão Screening of Marine Natural Products and Their Synthetic Derivatives for Antimicrobial and Antiproliferative Properties 17/2016 Mpindi John Patrick Bioinformatic Tools for Analysis, Mining and Modelling Large-Scale and Drug Testing Datasets 18/2016 Hilla Sumanen Work Disability among Young Employees Changes over Time and Socioeconomic Differences 19/2016 Oyediran Olulana Akinrinade Bioinformatic and Genomic Approaches to Study Cardiovascular Diseases 20/2016 Prasanna Sakha Development of Microfuidic Applications to Study the Role of Kainate Receptors in Synaptogenesis 21/2016 Neha Shrestha Mesoporous Silicon Systems for Oral Protein/Peptide-Based Diabetes Mellitus Therapy 22/2016 Tanja Holopainen Targeting Endothelial Tyrosine Kinase Pathways in Tumor Growth and Metastasis 23/2016 Jussi Leppilahti Variability of Gingival Crevicular Fluid Matrix Metalloproteinase -8 Levels in Respect to Point- of-Care Diagnostics in Periodontal Diseases 24/2016 Niina Markkula Prevalence, Predictors and Prognosis of Depressive Disorders in the General Population 25/2016 Katri Kallio FOLKHÄLSAN RESEARCH CENTER The Roles of Template RNA and Replication Proteins in the Formation of FACULTY OF MEDICINE Semliki Forest Virus Replication Spherules DOCTORAL PROGRAMME IN CLINICAL RESEARCH UNIVERSITY OF HELSINKI 26/2016

Helsinki 2016 ISSN 2342-3161 ISBN 978-951-51-2101-1 Folkhälsan Research Center Faculty of Medicine, University of Helsinki Heart and Lung Center, Helsinki University Hospital National Institute for Health and Welfare Finland

Doctoral Programme in Clinical Research

ROLE OF DEHYDROEPIANDROSTERONE IN HIGH-DENSITY LIPOPROTEIN-MEDIATED VASODILATION AND IN ADIPOSE TISSUE STEROID BIOSYNTHESIS

Hanna Paatela

ACADEMIC DISSERTATION

To be presented for public examination, with the permission of the Faculty of Medicine of the University of Helsinki, in lecture hall 2, Biomedicum Helsinki 1, Haartmaninkatu 8, Helsinki, on June 17th, 2016, at 12 noon.

Helsinki 2016 Supervised by Professor Matti J. Tikkanen, MD, PhD Folkhälsan Research Center University of Helsinki Heart and Lung Center, Helsinki University Hospital Helsinki, Finland

Adjunct Professor Matti Jauhiainen, PhD National Institute for Health and Welfare Genomics and Biomarkers Unit Helsinki, Finland

Reviewed by Professor Matti Poutanen, PhD Department of Physiology Institute of Biomedicine University of Turku Turku, Finland

Professor Raimo Voutilainen, MD, PhD Department of Pediatrics University of Eastern Finland and Kuopio University Hospital Kuopio, Finland

Official opponent Professor Oskari Heikinheimo, MD, PhD Obstetrics and Gynecology University of Helsinki and Helsinki University Hospital Helsinki, Finland

Dissertationes Scholae Doctoralis Ad Sanitatem Investigandam Universitatis Helsinkiensis 26/2016

ISBN 978-951-51-2101-1 (paperback) ISBN 978-951-51-2102-8 (PDF) ISSN 2342-3161 (print) ISSN 2342-317X (online) http://ethesis.helsinki.fi

Hansaprint Oy Turenki 2016

To my family

CONTENTS

List of original publications ...... 7 Abbreviations ...... 8 Abstract ...... 10 1 Introduction ...... 12 2 Review of the literature ...... 14 2.1 Overview of DHEA synthesis and ...... 14 2.2 Fatty acyl esters of DHEA and estradiol ...... 16 2.2.1 Formation and transport in blood ...... 16 2.2.2 Physiological role of steroid fatty acyl esters ...... 18 2.3 Effects of DHEA and estradiol on the cardiovascular system ...... 19 2.3.1 Epidemiology ...... 19 2.3.2 Effects of DHEA on the vascular wall ...... 20 2.3.3 Effects of estradiol on the vascular wall ...... 21 2.4 High-density lipoprotein ...... 22 2.4.1 General ...... 22 2.4.2 Antiatherogenic functions of HDL ...... 22 2.4.2.1 Vasodilatory effects of HDL ...... 23 2.5 Steroid hormone metabolism in adipose tissue ...... 24 2.5.1 Formation of and in adipose tissue ...... 24 2.5.2 Hydrolysis of steroid sulfates by steroid ...... 25 2.5.3 Other steroid-metabolizing ...... 27 2.5.4 Differences in steroid hormone metabolism between subcutaneous and visceral adipose tissue ...... 28 3 Aims of the study ...... 30 4 Materials and methods ...... 31 4.1 Subjects and samples ...... 31 4.1.1 Blood samples for incubations of with plasma ...... 31

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4.1.2 Umbilical cords for endothelial cell cultures ...... 31 4.1.3 Blood and adipose tissue samples for activity, mRNA expression, and hormone assays ...... 32 4.2 Methods ...... 32 4.2.1 Incubation of steroids with plasma ...... 32 4.2.1.1 Incorporation of DHEA in lipoproteins (Study I) ...... 32 4.2.1.2 Plasma incubation for cell culture experiments (Study II) ...... 33 4.2.1.3 Plasma incubation for arterial relaxation study (Studies I and II) ...... 33 4.2.1.4 Plasma incubation for endothelial nitric oxide synthase assay (unpublished) ...... 33 4.2.2 Isolation and purification of lipoproteins ...... 33 4.2.3 Thin-layer chromatography ...... 33 4.2.4 Arterial responses in vitro (Studies I and II) ...... 34 4.2.5 Endothelial nitric oxide synthase assay (unpublished) ...... 35 4.2.5.1 Cell culture ...... 35 4.2.5.2 Experimental design ...... 35 4.2.5.3 Western blot analysis ...... 36 4.2.6 Cellular uptake and hydrolysis of HDL-associated DHEA fatty acyl esters (Study II) ...... 36 4.2.7 Steroid sulfatase activity assay in adipose tissue (Study III) ...... 37 4.2.8 Quantitative real-time RT-PCR (Study III) ...... 37 4.2.9 Quantification of serum hormones (Study III) ...... 38 4.2.10 Other methods ...... 38 4.2.11 Statistical analyses ...... 38 5 Results ...... 39 5.1 Formation of DHEA fatty acyl esters in human plasma (Study I) ...... 39 5.2 Arterial responses in vitro (Studies I and II) ...... 40 5.2.1 Arterial responses to DHEA and estradiol ...... 40

5 5.2.2 Arterial responses to steroid fatty acyl ester-enriched HDL and native HDL ...... 40 5.2.3 Effect of blocking nitric oxide synthase and function of HDL, , and receptors ...... 41 5.3 Phosphorylation of endothelial nitric oxide synthase in endothelial cells (unpublished) ...... 43 5.4 Cellular uptake and hydrolysis of HDL-associated DHEA fatty acyl esters (Study II) ...... 44 5.5 Activity of steroid sulfatase and gene expression of steroid- converting enzymes in adipose tissue (Study III) ...... 47 5.5.1 Serum hormone concentrations ...... 47 5.5.2 Steroid sulfatase activity in adipose tissue ...... 47 5.5.3 Gene expression of steroid-converting enzymes in adipose tissue ...... 48 6 Discussion ...... 51 6.1 Incorporation of DHEA fatty acyl esters in plasma lipoproteins ...... 51 6.2 Vasodilatory effects of DHEA and estradiol ...... 51 6.3 Vascular effects of HDL-associated steroid fatty acyl esters ...... 52 6.3.1 Enhancement of vasodilatory effects of HDL ...... 52 6.3.2 Receptors involved in DHEA fatty acyl ester-induced vasodilation ...... 54 6.3.3 Role of endothelial nitric oxide synthase in DHEA fatty acyl ester-induced vasodilation ...... 55 6.3.4 Endothelial uptake and metabolism of HDL-associated DHEA fatty acyl esters ...... 56 6.4 DHEA metabolism in adipose tissue ...... 57 7 Summary, conclusions, and future perspective ...... 61 8 Acknowledgements ...... 63 9 References ...... 65

Original publications

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LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following publications, which are referred to in the text by their Roman numerals:

I Paatela H, Mervaala E, Deb S, Wähälä K, Tikkanen MJ. HDL- associated dehydroepiandrosterone fatty acyl esters: enhancement of vasodilatory effect of HDL. Steroids 2009;74:814-818.

II Paatela H, Vihma V, Jauhiainen M, Mervaala E, Tikkanen MJ. Dehydroepiandrosterone fatty acyl esters in high density lipoprotein: interaction with human vascular endothelial cells and vascular responses ex vivo. Steroids 2011;76:376-380.

III Paatela H, Wang F, Vihma V, Savolainen-Peltonen H, Mikkola TS, Turpeinen U, Hämäläinen E, Jauhiainen M, Tikkanen MJ. Steroid sulfatase activity in subcutaneous and visceral adipose tissue: a comparison between pre- and postmenopausal women. Eur J Endocrinol 2016;174:167-175.

In addition, some unpublished data are presented. The publications have been reprinted with the permission of their copyright holders (Studies I and II, Elsevier; Study III, BioScientifica Ltd.).

7 ABBREVIATIONS

ABCA1 ATP-binding cassette transporter A1 apoA-I apolipoprotein A-I BLT-1 blocker of lipid transport 1 (thiosemicarbazone, SR-BI inhibitor) CETP cholesteryl ester transfer protein CYP17A1 17α-hydroxylase/17,20-lyase CYP19A1 gene encoding DHEA dehydroepiandrosterone DHEAS dehydroepiandrosterone sulfate DTNB dithionitrobenzoic acid (LCAT inhibitor) E1 E1S E2 17β-estradiol eNOS endothelial nitric oxide synthase FAE fatty acyl ester FSH follicle-stimulating hormone HDL high-density lipoprotein HSD hydroxysteroid dehydrogenase HSD17B1 gene encoding 17β-hydroxysteroid dehydrogenase type 1 HSD17B7 gene encoding 17β-hydroxysteroid dehydrogenase type 7 HSD17B12 gene encoding 17β-hydroxysteroid dehydrogenase type 12 HSL hormone-sensitive HUVEC human umbilical vein endothelial cell IL-6 interleukin-6 IPO8 importin 8 KDM2B lysine (K)-specific demethylase 2B L-NAME nitro-L-arginine methyl ester (NOS antagonist) LCAT lecithin: acyltransferase LDL low-density lipoprotein LIPE gene encoding hormone-sensitive lipase MAPK mitogen-activated protein kinase (MAP kinase) mRNA messenger ribonucleic acid NOS nitric oxide synthase OAT organic anion transporter OATP organic anion transport polypeptide PCR polymerase chain reaction RT-PCR reverse transcription polymerase chain reaction S1P sphingosine-1-phosphate S1P3 sphingosine-1-phosphate receptor 3 SEM standard error of the mean SHBG -binding globulin

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SR-BI scavenger receptor class B, type I StAR steroidogenic acute regulatory protein STS steroid sulfatase STS gene encoding steroid sulfatase SULT SULT1E1 sulfotransferase that sulfates mainly estrogens (estrogen sulfotransferase) SULT2A1 sulfotransferase that sulfates non-aromatic steroids (DHEA sulfotransferase) TSPO translocator protein TLC thin-layer chromatography TNFα tumor necrosis factor α VLDL very low-density lipoprotein

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ABSTRACT

Background. Dehydroepiandrosterone (DHEA) and its sulfated form DHEA sulfate (DHEAS) are the most abundant steroid hormones in the circulation. These prohormones, secreted by the adrenal glands, are important precursors of biologically active androgens and estrogens. Adipose tissue is a major site for estrogen synthesis after menopause, when all estrogens are produced from hormone precursors in peripheral tissues. In the circulation, DHEA exists also as fatty acyl esters. These lipophilic derivatives of DHEA are transported by circulating lipoprotein particles and can be internalized and metabolized by peripheral tissues. DHEA and high- density lipoprotein (HDL) are both vascular relaxants. The aims were to study the role of DHEA fatty acyl esters in the HDL-mediated vasodilation, to study the cellular uptake and metabolism of HDL-associated DHEA fatty acyl esters in endothelial cells, and to investigate the metabolism of DHEAS in adipose tissue obtained from pre- and postmenopausal women. Methods. To incorporate DHEA or [3H]DHEA fatty acyl esters in lipoproteins, DHEA or [3H]DHEA was incubated with human plasma, in the presence and absence of lecithin:cholesterol acyltransferase (LCAT) inhibitor, and lipoproteins were isolated and purified. The radioactivity associated with lipoproteins was analyzed by thin-layer chromatography. Isolated rings of rat mesenteric arteries were precontracted with noradrenaline, and the relaxation responses were measured after the addition of increasing concentrations of native HDL, DHEA fatty acyl ester- enriched HDL, or HDL from the plasma incubation with DHEA and LCAT inhibitor. Selected experiments were carried out in rings pretreated with the inhibitors of endothelial nitric oxide synthase (eNOS), scavenger receptor class B, type I (SR-BI), androgen receptor, or estrogen receptors. Human umbilical vein endothelial cells were treated with [3H]DHEA fatty acyl ester-enriched HDL, and the radioactivity in the cellular and extracellular fractions was analyzed by thin-layer chromatography. The phosphorylation of eNOS in endothelial cells was analyzed by Western blot after treatment of the cells with DHEA fatty acyl ester-enriched HDL. In adipose tissue obtained from pre- and postmenopausal women, steroid sulfatase activity was measured as the conversion of [3H]DHEAS to [3H]DHEA, and mRNA expression levels of steroid-converting enzyme genes were determined by quantitative real-time RT-PCR. Results. DHEA became esterified and associated with lipoproteins during the incubation with plasma. The esterification was dependent on LCAT. DHEA fatty acyl ester-enriched HDL caused a dose-dependent arterial relaxation (mean, maximal 43%). This response was significantly stronger than the responses to native HDL (25%) and to HDL from the plasma incubation of DHEA in the presence of LCAT inhibitor (25%). Pretreatment

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of the arteries with eNOS antagonist attenuated the relaxation response to DHEA ester-enriched HDL (43% vs. 30%). Blocking of SR-BI almost completely abolished the relaxation response to DHEA ester-enriched HDL, whereas androgen and estrogen receptor blockage had no significant effect. In cultured endothelial cells, DHEA fatty acyl ester-enriched HDL caused a rapid phosphorylation of eNOS, which was ninefold compared to the effect of the buffer and did not differ from the effect of native HDL. When the cells were treated with [3H]DHEA fatty acyl ester-enriched HDL, intracellular radioactivity increased constantly during 24 h. The inhibition of SR-BI reduced this uptake by 30%. The proportion of unesterified [3H]DHEA increased intracellularly and in the culture media after several hours of treatment with [3H]DHEA ester-enriched HDL. Steroid sulfatase activity was higher in postmenopausal than in premenopausal women in abdominal subcutaneous (median 379 vs. 257 pmol DHEAS converted to DHEA/kg tissue/h) and visceral (545 vs. 360 pmol/kg/h) adipose tissue. Sulfatase activity was higher in visceral compared to subcutaneous fat in premenopausal and all women. The mRNA expression levels of three estradiol-producing enzymes in adipose tissue were higher in postmenopausal than in premenopausal women. The gene expression of steroid-converting enzymes varied between adipose tissue depots. Conclusions. DHEA fatty acyl ester-enriched HDL was a stronger vasodilator than native HDL, and this vascular relaxation was mediated by HDL receptor SR-BI. The esterification of DHEA was required for the improvement of the vasodilatory effect of HDL. Inhibition of eNOS significantly impaired the relaxation response to DHEA ester-enriched HDL, suggesting this response to be partly mediated by eNOS and nitric oxide. This is further supported by the finding in endothelial cells that DHEA ester- enriched HDL caused a rapid phosphorylation of eNOS, which is known to activate the enzyme. These results suggest that DHEA esters, cargos of HDL, may improve the vasodilatory and antiatherogenic functions of HDL. Human endothelial cells were able to internalize and hydrolyze HDL- associated DHEA fatty acyl esters and to further secrete the generated free DHEA from the cells. The cellular uptake was in part mediated by SR-BI. These results suggest that DHEA fatty acyl esters may act as hormone precursors transported to target tissues by lipoprotein particles. The cellular internalization and hydrolysis of DHEA esters were, however, slow processes and thus the liberated DHEA is presumably not responsible for the rapid vasodilatory effect of DHEA fatty acyl ester-enriched HDL. Steroid sulfatase activity in adipose tissue was higher in postmenopausal compared to premenopausal women, suggesting that circulating DHEAS could be more efficiently utilized by adipose tissue after menopause for the formation of biologically active sex hormones. Depot-differences in the steroid sulfatase activity and in the gene expression of steroid-converting enzymes suggest an important interplay between body fat distribution and steroid hormone metabolism.

11 Introduction

1 INTRODUCTION

Dehydroepiandrosterone (DHEA) and its sulfate ester dehydroepiandrosterone sulfate (DHEAS), secreted in large quantities by the adrenal glands, are important hormone precursors that can be converted to biologically active androgens and estrogens in peripheral tissues. In premenopausal women, the ovaries produce estrogens, but the contribution of peripheral tissues is significant, as well. At menopause, estrogen secretion by the ovaries completely ceases, and peripheral synthesis from adrenal precursors thus remains the only source of estrogens (Labrie 2015). DHEA fatty acyl esters are naturally occurring derivatives of DHEA, synthesized in blood and many tissues (Hochberg 1998). These derivatives are very lipophilic and thus almost exclusively incorporated in the lipoprotein particles in the circulation (Lavallée et al. 1996b). The biological role of lipoprotein-associated DHEA fatty acyl esters is unclear, but they may serve, after their cellular internalization and hydrolysis, as substrates for peripheral steroidogenesis (Vihma and Tikkanen 2011). DHEA and high- density lipoprotein (HDL) both improve endothelial function (Yuhanna et al. 2001, Simoncini et al. 2003). The role of HDL-associated DHEA fatty acyl esters in modulating vascular function is being explored in this thesis. In addition to circulating lipoproteins, DHEA may have relevance in another lipophilic environment — adipose tissue. Adipose tissue is a quantitatively important site of peripheral sex hormone biosynthesis. Adipose tissue expresses all the enzymes required for the formation of biologically active androgens and estrogens from adrenal prohormones (Li et al. 2015, Tchernof et al. 2015). Of these prohormones, DHEAS is the most abundant in the circulation and provides a large reservoir of substrate for the peripheral biosynthesis of active hormones. DHEAS is hydrophilic and thus needs an active transporter to cross cell membranes. After the transportation from the circulation to the adipose tissue, the first step in the formation of active sex hormones is the hydrolysis of DHEAS into DHEA, an action by steroid sulfatase. It has been shown that human adipocytes are able to internalize DHEAS, and subcutaneous adipose tissue as well as adipocytes can hydrolyze it (Dalla Valle et al. 2006). The capacity of adipose tissue to hydrolyze DHEAS has not been previously compared between premenopausal and postmenopausal women, or between subcutaneous and visceral adipose tissue depots. The role of DHEA in cardiovascular health remains controversial (Rice and Rees 2011). In the present doctoral thesis, we explored the role of DHEA in two different lipophilic environments — lipoproteins and adipose tissue. We investigated the possibility that lipophilic derivatives of DHEA incorporated in HDL particles could improve the vasodilatory capacity of HDL and therefore promote cardiovascular health. Furthermore, we

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investigated hydrolysis of DHEAS as well as gene expression of estrogen- producing enzymes in adipose tissue to elucidate the role of DHEA as a precursor of estrogens, which could influence vascular health especially in postmenopausal women.

13 Review of the literature

2 REVIEW OF THE LITERATURE

2.1 OVERVIEW OF DHEA SYNTHESIS AND METABOLISM

DHEA and its sulfate ester DHEAS are the most abundant steroid hormones in the circulation. DHEA and DHEAS are secreted primarily from the zona reticularis of the human adrenal cortex and to a lesser extent from the ovarian theca cells (Miller and Auchus 2011, Fritz and Speroff 2011). The pathway of DHEA synthesis from cholesterol is shown in Figure 1. Most of cholesterol supply in the adrenals comes from circulating low-density lipoproteins (LDL) by receptor-mediated endocytosis (Carr et al. 1980, Gwynne and Strauss 1982, Liu et al. 2000). To initiate steroidogenesis, cholesterol is mobilized from the outer to the inner mitochondrial membrane by the steroidogenic acute regulatory protein (StAR) (Clark et al. 1994) with the support of other outer mitochondrial membrane proteins, including translocator protein (TSPO) (Papadopoulos and Miller 2012). In the inner mitochondrial membrane, the cholesterol side-chain cleavage enzyme converts cholesterol to (Koritz and Kumar 1970, Miller and Auchus 2011). Pregnenolone is sequentially converted to 17-hydroxy- pregnenolone and then to DHEA by 17α-hydroxylase and 17, 20-lyase activities of the microsomal CYP17A1 enzyme. Sulfotransferase (SULT) enzyme SULT2A1 conjugates DHEA to form DHEAS (Miller and Auchus 2011). Secretion of DHEA and DHEAS by the adrenal glands increases during adrenarche at the age of 6–8 years. Maximal circulating concentrations of these prohormones are reached between the ages of 20 and 30 years, and thereafter the concentrations decline markedly during aging (Orentreich et al. 1984, Bélanger et al. 1994, Labrie et al. 1997b, Feldman et al. 2002). In the plasma, DHEA is predominantly bound to albumin (88%), and only a small proportion of DHEA is bound to sex hormone-binding globulin (SHBG) (8%) or exists in nonbound form (4%) (Fritz and Speroff 2011). DHEA and DHEAS are important hormone precursors that can be converted to biologically active sex steroids in peripheral tissues by the mechanisms of intracrinology (Labrie et al. 2005). The production of active androgens and estrogens from DHEA is outlined in Figure 1. DHEAS, which circulates in adult men and women at 1 000–100 000 times higher concentrations than 17β-estradiol (estradiol) and 100–5 000 times higher concentrations than testosterone (Labrie et al. 2011, Simpson 2003), provides a large reservoir of substrate for the conversion to active androgens and estrogens in peripheral tissues. In premenopausal women, estrogens are predominantly synthesized by the ovaries. After the cessation of ovarian estrogen synthesis at menopause, all estrogens and almost all androgens are

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Figure 1. Principal pathways of DHEA synthesis and metabolism. CYP17A1, 17α- hydroxylase/17,20-lyase; SULT, sulfotransferase; LCAT, lecithin:cholesterol acyltransferase; FAE, fatty acyl ester; HSL, hormone-sensitive lipase; STS, steroid sulfatase; HSD, hydroxysteroid dehydrogenase; E1, estrone; E2, 17β-estradiol; E1S, estrone sulfate. Not all the enzymes and metabolites are presented. synthesized in extragonadal sites through the enzymatic conversion from steroid precursors, DHEA and DHEAS (Labrie 2015). In postmenopausal women, approximately 80% of circulating DHEA is of adrenal origin, and 20% is secreted by the ovaries (Labrie et al. 2011). The conversion of steroid precursors to active sex hormones takes place in a large number of peripheral tissues, including adipose tissue, brain, mammary gland, prostate, skin, bone, and vascular endothelium and smooth muscle cells (Simpson 2003, Labrie et al. 2005, Tchernof et al. 2015). This peripheral conversion depends on the tissue-specific expression and activity of steroid-converting enzymes (Labrie et al. 2005, Li et al. 2015). The local sex steroid synthesis provides a possibility to control the formation and metabolism of hormones depending on the local requirements. Peripherally synthesized sex hormones mostly act at the site of their synthesis either in the same cell or tissue in a paracrine or intracrine fashion, and only a part is secreted to the circulation (Labrie et al. 1997a, Labrie 2015). In

15 Review of the literature postmenopausal women, concentrations of estrogens are higher in tissues than in serum (Badeau M et al. 2007, Yamatani et al. 2013, Savolainen- Peltonen et al. 2014, Kinoshita et al. 2014, Vihma et al. 2016), and serum levels of estrogens may reflect their production in tissues (Simpson 2003).

2.2 FATTY ACYL ESTERS OF DHEA AND ESTRADIOL

Steroid fatty acyl esters are naturally occurring lipophilic derivatives of steroid hormones. These derivatives, characterized as steroids bound to long- chain fatty acids by an ester bond, are synthesized in blood and several tissues. In the circulation, they are transported by lipoprotein particles (Hochberg 1998).

2.2.1 FORMATION AND TRANSPORT IN BLOOD Endogenous estradiol fatty acyl esters in human plasma were first described by Janocko and Hochberg (1983). Thereafter, lipophilic conjugates of DHEA, pregnenolone, and androst-5-ene-3β,17β-diol (androstenediol) were identified in human plasma, and it was demonstrated that these steroids as well as estradiol were converted to lipophilic conjugates when incubated with plasma (Jones and James 1985). In blood, the enzyme responsible for the fatty acyl esterification of DHEA, estradiol, pregnenolone, and androstenediol is lecithin:cholesterol acyltransferase (LCAT) (Roy and Bélanger 1989, Leszczynski et al. 1989, Leszczynski and Schafer 1991, Pahuja and Hochberg 1995, Lavallée et al. 1996a, Höckerstedt et al. 2002, Höckerstedt et al. 2004), the same enzyme that esterifies cholesterol (Figure 2). Of these steroid hormones, pregnenolone is the one that is most actively esterified in plasma, followed by DHEA and androstenediol. Estradiol is esterified in plasma at much lower rate than the other steroids (Jones and James 1985, Leszczynski and Schafer 1991, Pahuja and Hochberg 1995, Lavallée et al. 1996a). Similarly to cholesterol, DHEA, pregnenolone, and androstenediol are esterified in plasma at carbon 3 position, whereas estradiol is esterified only at carbon 17 (Leszczynski and Schafer 1991, Hochberg 1998, Kanji et al. 1999) (Figure 2). The fatty acid composition of biosynthetic and endogenous DHEA fatty acyl esters in plasma is similar to that of cholesteryl esters (Leszczynski and Schafer 1991, Pahuja and Hochberg 1995, Lavallée et al. 1996a). The esterification of DHEA and estradiol in blood takes place in HDL particles, specifically in HDL3 subfraction (Leszczynski et al. 1989, Leszczynski and Schafer 1991, Höckerstedt et al. 2002). The esterifying enzyme, LCAT, is associated with HDL particles. From these particles, DHEA and estradiol fatty acyl esters can be transferred to other lipoprotein particles, LDL and very low-density lipoprotein (VLDL) (Roy and Bélanger 1989, Helisten et al. 2001). The transfer of estradiol esters from HDL to other

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Figure 2. Formation of fatty acyl esters of DHEA, estradiol, and cholesterol in plasma. The esterification occurs on the surface of high-density lipoprotein (HDL) particles via the function of lecithin:cholesterol acyltransferase (LCAT). The fatty acyl esters formed are incorporated in the HDL particles. apoA-I, apolipoprotein A-I. lipoproteins is at least partly mediated by cholesteryl ester transfer protein (CETP) (Helisten et al. 2001), whereas the transfer of DHEA esters is CETP- independent (Provost et al. 1997). Fatty acyl esters of DHEA and estradiol are very lipophilic and thus almost exclusively associated with lipoprotein particles in the circulation (Larner et al. 1987, Roy and Bélanger 1989, Roy and Bélanger 1993, Lavallée et al. 1996b, Vihma et al. 2003a). DHEA fatty acyl esters circulate in humans at nanomolar concentrations (Labrie et al. 1997b, Wang F et al. 2011), whereas estradiol ester concentrations are picomolar (Vihma et al. 2003b, Badeau M et al. 2007, Savolainen-Peltonen et al. 2014). During aging, DHEA fatty acyl ester levels in serum in both men and women decline markedly, although not as dramatically as DHEA and DHEAS concentrations (Bélanger et al. 1994, Labrie et al. 1997b). In the circulation, a significant proportion of DHEA is in fatty acylated form. In earlier studies with immunoassay-based analytical methods for hormone quantification, the concentration of DHEA fatty acyl esters in male and female serum has been approximately half that of unconjugated DHEA (Roy and Bélanger 1993, Bélanger et al. 1994, Labrie et al. 1997b). More recently, analyzed with mass spectrometric method from female serum, the mean proportion of DHEA fatty acyl esters of total DHEA was 9% in premenopausal and 11% in postmenopausal women (Wang F et al. 2011).

17 Review of the literature

2.2.2 PHYSIOLOGICAL ROLE OF STEROID FATTY ACYL ESTERS The biological role of steroid fatty acyl esters remains still somewhat unclear. Lipoprotein-associated steroid fatty acyl esters, after internalization to target cells, may play a role in peripheral steroidogenesis. Pregnenolone fatty acyl esters in lipoprotein particles are internalized by porcine ovarian granulosa cells and hydrolyzed to pregnenolone, which is further converted to progesterone and other metabolites (Roy and Bélanger 1991, Roy and Bélanger 1992). Similarly, bovine and guinea pig adrenocortical cells can internalize and convert lipoprotein-associated pregnenolone fatty acyl esters to unconjugated steroids (Provencher et al. 1992). LDL-associated DHEA fatty acyl esters are internalized by ZR-75-1 human cells and further converted to DHEA and androstenediol (Roy and Bélanger 1993). Wang F. and co-workers (2008) showed in cultured human HeLa cells that cellular uptake of LDL-associated DHEA fatty acyl esters was mediated by LDL receptors or LDL receptor-related receptors, and after the internalization, DHEA esters were hydrolyzed and metabolized, followed by the secretion of the metabolites from the cells. These studies suggest that lipoprotein-associated fatty acyl esters of hormone precursors may be transferred to peripheral cells where they can serve as substrates for the biosynthesis of active hormones. In addition to DHEA and pregnenolone, lipoprotein-associated estradiol may be internalized by target cells. Gong and co-workers (2003) demonstrated that endothelial cells were able to internalize HDL-associated estradiol via scavenger receptor class B, type I (SR-BI), and that HDL- associated estradiol stimulated endothelial nitric oxide synthase (eNOS). Another study showed that osteoblastic cells were able to internalize radiolabeled estradiol associated with LDL and HDL3 particles through SR-B receptors (Brodeur et al. 2008). These two studies, however, did not characterize the molecular form of the lipoprotein-bound estradiol, and the authors did not discuss the possibility that estradiol could be in fatty acyl esterified form (Badeau M et al. 2008). Rat hepatoma cells have been demonstrated to rapidly internalize HDL-incorporated estradiol fatty acyl esters. Both SR-BI and LDL receptors were involved in this cellular uptake. Furthermore, the proportion of esterified estradiol decreased and that of free estradiol increased intracellularly during a 24-h incubation, indicating that estradiol esters were hydrolyzed in the cells to biologically active estradiol (Badeau RM et al. 2007). Steroid fatty acyl esters may represent a storage form of hormones. In adipose tissue, the concentration of estradiol fatty acyl esters is higher than that in serum (Larner et al. 1992, Badeau M et al. 2007, Wang F et al. 2013, Savolainen-Peltonen et al. 2014). Adipose tissue has a high capacity to fatty acyl esterify estradiol (Kanji et al. 1999, Wang F et al. 2012). De-esterifying enzymatic activity is present, as well (Wang F et al. 2012). As fatty acyl esterified estradiol is inactive and unable to bind to estrogen receptors (Janocko et al. 1984), the balance between the esterifying and hydrolyzing

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capacities may play an important role in the activating and inactivating processes of estradiol in tissues. Lipoprotein-incorporated estradiol fatty acyl esters have been proposed to protect lipoproteins against oxidation (Shwaery et al. 1997, Shwaery et al. 1998) and therefore to have antiatherogenic potential. In vitro, esterified estradiol in lipoproteins can, however, increase the resistance of lipoproteins against oxidation only at high supraphysiological concentrations (Meng et al. 1999, Höckerstedt et al. 2004). Clinical studies on the effect of estrogen therapy against LDL oxidation have produced conflicting results and have not been able to confirm the antioxidative protection by estrogens (Vihma and Tikkanen 2011).

2.3 EFFECTS OF DHEA AND ESTRADIOL ON THE CARDIOVASCULAR SYSTEM

2.3.1 EPIDEMIOLOGY The dramatic age-related decline in circulating DHEA and DHEAS levels has been proposed to be related with the development of several diseases associated with aging, including cardiovascular diseases (Tchernof and Labrie 2004). Studies addressing the association between DHEA and cardiovascular diseases have produced somewhat inconsistent findings (Tchernof and Labrie 2004, Savineau et al. 2013, Ohlsson et al. 2015). Several epidemiological studies indicate that low circulating DHEA and DHEAS levels are associated with increased risk for cardiovascular disease outcomes in men (Barrett-Connor et al. 1986, Trivedi and Khaw 2001, Ohlsson et al. 2010, Tivesten et al. 2014), but some studies have found no association (Tilvis et al. 1999, Haring et al. 2013). Although the literature is inconsistent, available evidence suggests an association between low DHEA or DHEAS concentration and increased all-cause and cardiovascular mortality in men (Ohlsson et al. 2015), whereas in women there may be a U- shaped association or no association (Tchernof and Labrie 2004, Ohlsson et al. 2015). The lower incidence of cardiovascular disease in premenopausal women compared to age-matched men and the increased risk for cardiovascular outcomes after the menopause have led to the suggestion that premenopausal women are protected from cardiovascular disease by endogenous estrogens (Vitale et al. 2009). Cardiovascular effects of postmenopausal hormone therapy are, however, controversial (Hale and Shufelt 2015). Several observational studies indicate a protective role of postmenopausal hormone therapy against coronary heart disease (Grodstein et al. 1999, Grodstein et al. 2000, Kim et al. 2006). Randomized placebo- controlled trials, however, have failed to confirm cardiovascular protection by hormone therapy (Hulley et al. 1998, Rossouw et al. 2002). Age or time

19 Review of the literature since menopause at the initiation of hormone therapy may be important factors for modulating the cardiovascular effects. The evidence is supporting a beneficial effect of hormone therapy on cardiovascular health when started early after menopause (Mikkola and Clarkson 2002, Tuomikoski and Mikkola 2014).

2.3.2 EFFECTS OF DHEA ON THE VASCULAR WALL Endothelial dysfunction, characterized as decreased bioavailability of nitric oxide, is associated with the development of cardiovascular diseases (Cockcroft 2005). Nitric oxide, synthesized in the vascular wall by eNOS, is a powerful vasodilatory, anti-inflammatory, and antiatherogenic agent (Landmesser et al. 2004). There is evidence that steroid hormones regulate vascular function. In addition to genomic effects, sex steroids have rapid, nongenomic actions on the vascular endothelium (Chow et al. 2010). Although DHEA mostly serves as a substrate for the synthesis of biologically active sex hormones, it also directly acts on the vascular endothelium. In cultured human endothelial cells, DHEA increases the synthesis of nitric oxide by transcriptional induction of eNOS expression as well as through rapid, nongenomic activation of eNOS (Simoncini et al. 2003). These effects of DHEA are not mediated through conversion to androgens or estrogens (Simoncini et al. 2003). Two other studies have demonstrated that the rapid activation of eNOS and increased synthesis of nitric oxide by DHEA are mediated through a specific G-protein-coupled plasma membrane receptor (Liu and Dillon 2002, Liu and Dillon 2004). The possible membrane receptor, however, remains unidentified (Simoncini and Genazzani 2007). Exact cellular mechanisms by which DHEA rapidly activates eNOS are not fully understood. Signaling through mitogen- activated protein kinase (MAPK) or phosphatidylinositol-3-kinase/Akt kinase pathways has been suggested (Simoncini et al. 2003, Liu and Dillon 2004, Formoso et al. 2006, Bhuiyan et al. 2011). In ovariectomized rats, DHEA treatment has been shown to increase the phosphorylation of eNOS and to improve endothelium-dependent vasodilation in aortas isolated after the treatment (Camporez et al. 2011). In ovariectomized rabbits with high-cholesterol diet, DHEA treatment enhanced endothelium-dependent vasodilation and nitric oxide release in isolated aortas, and these effects were attenuated by a simultaneous treatment with aromatase inhibitor, suggesting that the effects of DHEA were partly mediated through conversion to estrogens (Hayashi et al. 2000). Human studies in vivo have shown that serum DHEAS concentration correlates positively with flow-mediated dilation of brachial artery (Akishita et al. 2008) and negatively with carotid intima-media thickness (Meyer et al. 2005, Yoshida et al. 2010, Mieczkowska et al. 2012), a surrogate measure of atherosclerosis. Only few clinical trials of DHEA therapy on vascular function have been conducted in humans. In a placebo-controlled study in 24

20

hypercholesterolemic men, a short-term (12 weeks) DHEA supplementation improved endothelium-dependent flow-mediated dilation of the brachial artery, thus improving endothelial function (Kawano et al. 2003). Similarly, in 36 postmenopausal women, DHEA treatment increased flow-mediated dilation of the brachial artery and reduced serum total cholesterol concentrations compared to the placebo group (Williams et al. 2004). However, a study in 40 patients with adrenal insufficiency failed to show any effect of 12-week DHEA treatment on endothelial function (Rice et al. 2009). Furthermore, in a small study in 16 postmenopausal women, a four-week DHEA supplementation failed to improve flow-mediated dilation, whereas hormone therapy (estrogen and progestin) improved endothelial function (Silvestri et al. 2005). In addition to stimulating the nitric oxide synthesis and vasodilation, DHEA may improve vascular function through other mechanisms. In vitro, DHEA stimulates endothelial proliferation and angiogenesis (Liu et al. 2008), protects endothelial cells against apoptosis (Liu et al. 2007), and regulates the synthesis of vasoconstrictor endothelin-1 (Chen et al. 2008).

2.3.3 EFFECTS OF ESTRADIOL ON THE VASCULAR WALL There is a large body of evidence that estrogens regulate vascular function (Mendelsohn and Karas 1999, Simoncini et al. 2006, Chakrabarti et al. 2014). Estradiol rapidly causes vasodilation in coronary and peripheral arteries (Williams et al. 1992, Gilligan et al. 1994, Guetta et al. 1997). In endothelial cells, estradiol increases eNOS expression (Kleinert et al. 1998), and also rapidly activates eNOS (Lantin-Hermoso et al. 1997, Chen et al. 1999), thus stimulating the synthesis of nitric oxide (Chambliss and Shaul 2002). The rapid, nongenomic effects of estradiol on the endothelium are mediated through membrane-bound estrogen receptors (Chen et al. 1999, Simoncini et al. 2000). Classically estrogens act through nuclear estrogen receptors regulating gene expression. Estrogen receptors, however, also function outside the nucleus, and ligand binding to these cell membrane receptors launches several intracellular signal cascades (Kim et al. 2008, Banerjee et al. 2014). On endothelial cell plasma membrane, estrogen receptor α and eNOS are both located in caveolae, domains enriched with cholesterol and sphingomyelin (Chambliss et al. 2000). In endothelial cells, estradiol treatment enhances, via estrogen receptor, the eNOS phosphorylation, which activates the enzyme leading to the secretion of nitric oxide (Chen et al. 1999, Wyckoff et al. 2001, Florian et al. 2004).

21 Review of the literature

2.4 HIGH-DENSITY LIPOPROTEIN

2.4.1 GENERAL The biogenesis of HDL starts with the secretion of apolipoprotein A-I (apoA-I) by the liver and the intestine. Secreted apoA-I acquires phospholipids and a small amount of cholesterol through interaction with ATP-binding cassette transporter A1 (ABCA1) resulting in the formation of nascent preβ-HDL particles. They are converted to discoidal HDL when acquiring additional cholesterol and phospholipids from peripheral cells. Discoidal HDL particles are gradually transformed into spherical particles via the LCAT function whereby free cholesterol is converted to cholesteryl esters, which form the lipophilic core of the mature HDL particle (Zannis et al. 2015). Plasma HDL particles are a heterogeneous group of particles differing in size, shape, charge, and composition. HDL particles transport lipids, proteins, vitamins, steroid hormones, different metabolites, and small RNAs. These numerous cargos of HDL are transported between the tissue sites and are important for the functionality of HDL (Vickers and Remaley 2014).

2.4.2 ANTIATHEROGENIC FUNCTIONS OF HDL Low plasma HDL cholesterol concentration is a strong risk factor for cardiovascular disease. Epidemiological studies indicate that the HDL cholesterol level in blood is inversely correlated with the risk for cardiovascular disease (Miller and Miller 1975, Gordon et al. 1977). However, recent clinical trials aiming at rising HDL cholesterol have failed to reduce cardiovascular events (Barter et al. 2007, AIM-HIGH Investigators 2011, Schwartz et al. 2012). In addition to these clinical trials, studies of human genetics have failed to support the conventional HDL cholesterol hypothesis (Edmondson et al. 2009, Voight et al. 2012). These findings have led to the understanding of the complexity of HDL biology. The HDL-mediated atheroprotection may be related to the functionality of HDL, and not necessarily to elevated HDL cholesterol levels (Vickers and Remaley 2014, Kontush 2014). HDL particles have several atheroprotective activities, which include reverse cholesterol transport, vasodilatory, cytoprotective, anti- inflammatory, antioxidative, and antithrombotic actions as well as regulating gene expression by microRNAs and improving glucose metabolism (Kontush 2014). The clinical relevance and importance of these mechanisms are still partly unclear. The best studied and most important biological activity of HDL is the function in the reverse cholesterol transport process, i.e. removal of cholesterol from peripheral tissues and transport to the liver for excretion. HDL particles acquire cholesterol from arterial walls, primarily from macrophages and macrophage-derived foam cells. The esterified cholesterol

22

in HDL particles is taken directly to the liver and steroidogenic tissues by SR-BI-mediated selective uptake or transferred by CETP to VLDL and LDL particles, which are eventually delivered to the liver by LDL receptors. From the liver, cholesterol is used for synthesis or directly excreted into the bile (Lewis and Rader 2005).

2.4.2.1 Vasodilatory effects of HDL In addition to its role in the reverse cholesterol transport process, HDL directly acts on the vascular endothelium, thus influencing cardiovascular health. On the endothelium, HDL has vasodilatory activity, which is primarily mediated through nitric oxide release by endothelial cells (Yuhanna et al. 2001, Nofer et al. 2004) but also through production of prostacyclin (Norata et al. 2004, Zhang et al. 2012). HDL stimulates eNOS activity in cultured endothelial cells and causes endothelium-dependent vasodilation in isolated mouse aortas (Yuhanna et al. 2001). These effects are mediated through HDL binding to SR-BI (Yuhanna et al. 2001, Li et al. 2002), which is expressed in endothelium and highly enriched in caveolae (Uittenbogaard et al. 2000, Yuhanna et al. 2001). In addition to SR-BI, sphingosine-1-phosphate (S1P) receptors are involved in eNOS activation by HDL (Nofer et al. 2004, Kimura et al. 2006). S1P, a cargo of HDL, binds to apolipoprotein M in HDL and mediates vasculoprotective actions through S1P receptors on the endothelium (Christoffersen et al. 2011, Karuna et al. 2011). S1P has been shown to cause vasodilation in aortic rings of mice and rats (Nofer et al. 2004, Roviezzo et al. 2006). In aortas from mice lacking the S1P3 receptor, the vasodilatory effect of HDL is partly attenuated (Nofer et al. 2004). The induction of nitric oxide and prostacyclin by HDL-associated S1P are of importance in the regulation of many cellular events, including proliferation, apoptosis, cell adhesiveness, angiogenesis, and vascular integrity (Rodríguez et al. 2009). Both SR-BI and S1P receptors are involved in the vasculoprotective effects of HDL (Rodríguez et al. 2009, Mineo and Shaul 2012). The activity of eNOS is regulated by complex signal transduction pathways, involving activation of several kinases leading to changes in the phosphorylation state of eNOS (Mineo and Shaul 2013). HDL stimulates phosphorylation of Akt kinase, which in turn phosphorylates eNOS at serine-1177. HDL also causes parallel activation of the MAP kinase pathway, and these two pathways lead to the activation of the eNOS enzyme (Mineo et al. 2003). HDL enhances the production of nitric oxide and subsequent vasorelaxation by several other mechanisms than modulating eNOS activity. HDL increases the half-life of eNOS protein and thus the abundance of the enzyme (Rämet et al. 2003). HDL preserves, in the presence of oxidized LDL, the lipid environment in the caveolae, and thereby maintains the subcellular localization and normal function of eNOS (Uittenbogaard et al. 2000).

23 Review of the literature

Several studies in humans have demonstrated the relationship between HDL cholesterol levels and endothelium-dependent vasodilation. Elevated serum HDL cholesterol ameliorates the abnormal endothelium-dependent vasodilation in coronary arteries in patients with early stage of coronary atherosclerosis (Zeiher et al. 1994). Furthermore, HDL cholesterol levels correlate positively with flow-mediated dilation of the brachial artery (Li et al. 2000, Kuvin et al. 2003). Steroid hormones, also cargos of HDL, may play a role in vasodilation caused by the lipoprotein. Gong and co-workers (2003) demonstrated that HDL delivered estradiol to endothelial cells, and HDL isolated from women had a significantly higher capacity in endothelial cells to activate eNOS than had HDL from men, suggesting that HDL-associated estradiol could mediate eNOS activation by HDL. Another study, however, found no difference between the relaxation responses to female HDL and male HDL in isolated mouse aortic rings (Nofer et al. 2004). In vivo in humans, change in serum estradiol fatty acyl ester concentration during postmenopausal estrogen treatment correlated positively with enhanced forearm blood flow responses (Vihma et al. 2003b). Steroid fatty acyl esters, such as DHEA and estradiol esters, are associated with HDL particles in the circulation. The possible role of HDL-associated DHEA and estradiol fatty acyl esters in HDL-mediated eNOS activation and vasodilation has not been previously studied.

2.5 STEROID HORMONE METABOLISM IN ADIPOSE TISSUE

Adipose tissue is not only an energy storage, but rather an active endocrine and metabolic organ that contributes to the metabolic homeostasis by secreting and metabolizing a large variety of adipokines and hormones (Kershaw and Flier 2004). Accumulation of visceral adipose tissue is associated with metabolic alterations that increase the risk for metabolic syndrome, type 2 diabetes, and cardiovascular disease (Fox et al. 2007, Smith et al. 2012). Sex hormones are important regulators of body fat distribution patterns (Tchernof and Després 2013).

2.5.1 FORMATION OF ANDROGENS AND ESTROGENS IN ADIPOSE TISSUE Adipose tissue is an active endocrine organ and an important site for extragonadal steroid hormone biosynthesis and metabolism. This is supported by the findings that the concentrations of DHEA, androstenedione, estradiol, and estrone are higher in adipose tissue than in blood (Deslypere et al. 1985, Badeau M et al. 2007, Wang F et al. 2011, Savolainen-Peltonen et al. 2014, Kinoshita et al. 2014, Vihma et al. 2016). Adipose tissue contains all the necessary enzyme machinery for the synthesis

24

of biologically active androgens and estrogens from the precursors, DHEA and DHEAS (Tchernof et al. 2015, Li et al. 2015) (Figure 1). In premenopausal women, estrogens are secreted by the ovaries, but the extragonadal synthesis represents a significant source, as well (Labrie et al. 2003). In postmenopausal women and in men, extragonadal synthesis is the only source of estrogens (Labrie et al. 2003, Simpson 2003, Labrie 2015). Adipose tissue is one of the largest endocrine tissues and thus quantitatively important in the formation of active sex hormones. The concentration of DHEA in adipose tissue in women is approximately ten times higher than that in serum (Deslypere et al. 1985, Wang F et al. 2011, Kinoshita et al. 2014), suggesting that DHEA and DHEAS are effectively transferred from the circulation to the adipose tissue. It is uncertain whether adipose tissue is able to generate de novo sex hormones directly from cholesterol. Gene expression of StAR, a protein required for the delivery of cholesterol to the inner mitochondrial membrane, has been detected at low levels in subcutaneous and visceral adipose tissue obtained from pregnant women undergoing caesarean section (MacKenzie et al. 2008) as well as from obese male and female subjects (Hernandez-Morante et al. 2009). Gene expression of cholesterol side-chain cleavage enzyme has also been found in both adipose tissue depots (MacKenzie et al. 2008). The activity of this enzyme in adipose tissue is, however, unknown. In subcutaneous adipose tissue of women, 17α- hydroxylase activity of CYP17A1, measured as conversion of progesterone to 17α-hydroxyprogesterone, has been detected (Puche et al. 2002). Furthermore, both 17α-hydroxylase and 17, 20-lyase activities of CYP17A1 have been found in human subcutaneous and visceral fat (Kinoshita et al. 2014). Other studies, however, failed to detect mRNA expression of CYP17A1 in adipose tissue (Dalla Valle et al. 2006, MacKenzie et al. 2008, Wang L et al. 2012). In the study by Dalla Valle and co-workers (2006), mRNA expression of any of these key proteins and enzymes, namely StAR, cholesterol side-chain cleavage enzyme, and CYP17A1, could not be detected in human subcutaneous adipose tissue. Thus, the ability of adipose tissue to synthesize active steroid hormones from cholesterol remains uncertain, whereas the capacity of adipose tissue acting as a major steroid-converting site is well established.

2.5.2 HYDROLYSIS OF STEROID SULFATES BY STEROID SULFATASE Sulfated steroids have been considered as inactive metabolic end products that are water-soluble and thus secreted to the urine. However, over the past two decades, extensive research indicates that inactive steroid sulfates are important sources of active steroid hormones. DHEAS circulates at micromolar concentration, and thus provides a large reservoir of substrate for peripheral steroidogenesis. Estrone sulfate is the most abundant

25 Review of the literature circulating estrogen in postmenopausal women (Labrie et al. 2011). DHEAS and estrone sulfate have prolonged half-lives in plasma compared to unconjugated DHEA and estrone (Ruder et al. 1972, Kroboth et al. 1999). High plasma concentrations of DHEAS and estrone sulfate together with their long half-lives have given rise to the view that these steroid conjugates could act as reservoirs of substrate for the formation of active hormones (Reed et al. 2005, Mueller et al. 2015). Sulfation and desulfation of steroid hormones represent an important mechanism in regulating steroidogenesis and hormone action in tissues (Mueller et al. 2015). DHEAS and estrone sulfate are hydrophilic and thus unable to cross cell membranes. They require active transmembrane transport for cellular uptake. This uptake is performed by two organic anion carrier protein families: organic anion transport polypeptides (OATPs) (Hagenbuch and Stieger 2013) and organic anion transporters (OATs) (Koepsell 2013). These proteins are expressed in several tissues and possess distinct substrate specificity and uptake kinetics for different conjugates (Mueller et al. 2015). Primary transporters of sulfated steroids appear to be the OATPs (Mueller et al. 2015), but also some OATs can transport steroid sulfates, as shown in human placenta (Cha et al. 2000). Dalla Valle and co- workers (2006) found that in human subcutaneous adipose tissue, genes encoding three OATPs, namely OATP-B, OATP-D, and OATP-E, were expressed, while other members of the OATP and OAT families were not. Furthermore, transmembrane transport of DHEAS was observed in cultured preadipocytes and mature adipocytes (Dalla Valle et al. 2006). Once internalized, DHEAS and estrone sulfate must be hydrolyzed before being further converted to active steroid hormones. Steroid sulfatase, a membrane-bound microsomal enzyme, catalyzes desulfation of its two major substrates DHEAS and estrone sulfate to DHEA and estrone, respectively (Reed et al. 2005, Purohit et al. 2011, Mueller et al. 2015). Steroid sulfatase activity was first demonstrated in rat liver microsomes by Dodgson and co- workers (1954). Since then, steroid sulfatase has been found in several tissues, such as adrenals, prostate, breast, ovary, testis, skin, brain, bone, thyroid, pancreas, and endometrium, which all show steroid sulfatase activity (Mueller et al. 2015). In adipose tissue, Martel and co-workers (1994) were the first to show significant DHEA and estrone sulfatase activity in mesenteric fat of rhesus monkey. In human subcutaneous adipose tissue, the gene and protein expression as well as activity of steroid sulfatase in the conversion of DHEAS to DHEA have been detected (Dalla Valle et al. 2006). Furthermore, preadipocytes and adipocytes are able to hydrolyze DHEAS (Dalla Valle et al. 2006). In human visceral adipose tissue, steroid sulfatase activity has not been previously studied. However, mRNA expression of steroid sulfatase gene (STS) has been detected both in subcutaneous and in visceral adipose tissue depots (Blouin et al. 2009a, Wang F et al. 2013). Relatively little is known about the regulation of steroid sulfatase expression and activity. The STS gene displays alternative promoter usage

26

and splicing, which is thought to be the basis for the tissue-specific regulation (Mueller et al. 2015). In adipose tissue, transcriptional regulation of steroid sulfatase seems to differ from that of placental steroid sulfatase (Dalla Valle et al. 2006). Steroid sulfatase can be controlled via posttranscriptional and posttranslational modifications, such as glycosylation (Stein et al. 1989). Breast cancer tissue displays high estrone sulfatase activity (Santner et al. 1984, Pasqualini et al. 1996), and the mRNA expression levels of STS are higher in cancer compared to normal tissue (Utsumi et al. 2000, Honma et al. 2006), suggesting an important role of steroid sulfatase in hormone-dependent tumor growth (Purohit and Foster 2012). The exact mechanisms stimulating steroid sulfatase expression and activity in breast cancer are somewhat unclear. In breast cancer cells, STS mRNA expression is upregulated by estradiol (Evans et al. 1993, Zaichuk et al. 2007). Inflammatory cytokines, such as tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and IL-1β, increase estrone sulfatase activity in breast cancer cells (Purohit et al. 1996, Newman et al. 2000, Honma et al. 2002). The enhanced activity of steroid sulfatase by IL-6 and TNFα was observed without an increase in STS mRNA expression, suggesting a posttranslational effect (Newman et al. 2000). In hepatic cells, inflammatory stimuli induce gene expression and activity of steroid sulfatase, thus activating estrogens, which in return attenuate the inflammation via the estrogen receptor signaling (Jiang et al. 2016). In adipose tissue, the regulation of steroid sulfatase activity is largely unknown.

2.5.3 OTHER STEROID-METABOLIZING ENZYMES DHEA, derived from the circulation or from desulfation of DHEAS in the peripheral tissue, can be further converted to active sex steroids by sequential action of steroid-converting enzymes. The classical pathway of the production of androgens and estrogens from DHEA is shown in Figure 1. In addition to this classical pathway, there may be other enzymes and pathways involved in steroid biosynthesis in peripheral tissues. These alternative pathways may have relevance in peripheral steroidogenesis and especially in the local production of active sex steroids in cancerous tissues, such as (Fokidis et al. 2015). In adipose tissue and adipocytes, DHEA can be reduced to androstenediol (Schindler and Aymar 1975, Marwah et al. 2006) in a reaction catalyzed by 17β-hydroxysteroid dehydrogenase (17βHSD) enzymes (Tchernof et al. 2015). Androstenediol, although structurally an androgen, binds to estrogen receptors (Kuiper et al. 1997) and has estrogenic properties, such as stimulation of breast cancer cell proliferation in an estrogen receptor- dependent manner (Poulin and Labrie 1986, Purohit et al. 2011). In cultured stromal cells from breast adipose tissue, DHEA is converted to androstenedione and estrone, indicating 3βHSD and aromatase activities (Killinger et al. 1995). Aromatase activity, in the conversion of

27 Review of the literature androstenedione to estrone (Nimrod and Ryan 1975, Forney et al. 1981, Deslypere et al. 1985) and testosterone to estradiol (Nimrod and Ryan 1975, Forney et al. 1981), has been demonstrated in adipose tissue. Estrone can be reduced to biologically active estradiol by 17βHSDs in adipose tissue (Martel et al. 1992) and adipocytes (Bellemare et al. 2009). Of the enzyme isoforms of 17βHSD, types 1, 7, and 12 are known to convert estrone to estradiol (Saloniemi et al. 2012, Tchernof et al. 2015). It is unclear, however, which of these isoenzymes are responsible for the conversion of estrone to estradiol in adipose tissue. In human adipocytes, the mRNA expression of type 12 was higher compared to types 1 and 7, suggesting that 17βHSD type 12 may act as one of the enzymes converting estrone to estradiol in adipose tissue (Bellemare et al. 2009, Tchernof et al. 2015). Based on studies in genetically modified mouse models, 17βHSD enzymes have, in addition to their role in sex steroid metabolism, important roles in other metabolic processes (Saloniemi et al. 2012). Type 7 isoform of 17βHSD is essential for cholesterol biosynthesis in mice, and type 12 is involved in the elongation of essential fatty acids, such as arachidonic acid, thus influencing prostaglandin synthesis. In adipocytes derived from adipose tissue of men (Blouin et al. 2006) and women (Blouin et al. 2003, Blouin et al. 2009a), androstenedione was reduced to testosterone by 17βHSD activity and also to a more potent androgen, dihydrotestosterone, indicating the presence of 5α-reductase activity. In addition to the classical pathway proceeding through DHEA and testosterone, dihydrotestosterone is proposed to be synthesized by an alternative pathway, also called “backdoor” pathway, in which dihydrotestosterone is produced from an inactive steroid, 3α-androstanediol (Auchus 2004, Luu-The 2013). The existence of this pathway has not been described in adipose tissue. Hormone-sensitive lipase, an enzyme participating in the hydrolysis of triacylglycerols in adipose tissue (Holm 2003), is able to hydrolyze fatty acyl esters of DHEA and is partly responsible for the hydrolysis of estradiol fatty acyl esters in adipose tissue (Wang F et al. 2012). In adipose tissue in women, a significant proportion of estradiol is in inactive fatty acyl esterified form (Larner et al. 1992, Badeau M et al. 2007, Wang F et al. 2013, Savolainen-Peltonen et al. 2014), whereas DHEA fatty acyl esters have not been detected (Wang F et al. 2011). DHEA esters are more efficiently hydrolyzed compared to estradiol esters in adipose tissue in vitro (Wang F et al. 2012).

2.5.4 DIFFERENCES IN STEROID HORMONE METABOLISM BETWEEN SUBCUTANEOUS AND VISCERAL ADIPOSE TISSUE Sex steroids are important regulators of adiposity and body fat distribution. Body fat distribution differs between men and women. Men and postmenopausal women usually have increased visceral adiposity, whereas

28

premenopausal women tend to have increased subcutaneous fat accumulation (Tchernof and Després 2013, Palmer and Clegg 2015). Estrogen receptors α and β are present in adipose tissue, supporting the direct estrogenic action in adipose tissue (Mizutani et al. 1994, Crandall et al. 1998, Dieudonné et al. 2004). Reduced estrogen levels in postmenopausal women have been associated with increased visceral fat accumulation after menopause (Genazzani and Gambacciani 2006, Lovejoy et al. 2008). Steroid hormone metabolism appears to be somewhat different between adipose tissue depots. In obese men, androgen concentrations differ between abdominal subcutaneous and visceral adipose tissues and associate positively with lipolytic responsiveness in omental adipocytes suggesting a regulatory effect of androgens in this depot (Bélanger et al. 2006). Several steroid- converting enzyme genes display different expression levels in subcutaneous and visceral adipose tissue depots. Steroid sulfatase gene is more expressed in subcutaneous than in visceral adipose tissue in women (Blouin et al. 2009a, Wang F et al. 2013) and in obese men (Wang F et al. 2013). In preadipocytes derived from female omental adipose tissue, the STS mRNA expression was higher than in preadipocytes from subcutaneous fat, and following adipocyte differentiation, the STS expression increased (Blouin et al. 2009a). These findings suggest that the differences in the STS expression between adipose tissue depots may be due to the relative proportion of mature adipocytes and preadipocytes in each depot (Blouin et al. 2009a). In addition to steroid sulfatase, the mRNA expression levels of aromatase and hormone-sensitive lipase seem to be higher in subcutaneous than in visceral adipose tissue (Reynisdottir et al. 1997, Blouin et al. 2006, Ray et al. 2009, Wang F et al. 2013). Other studies, however, have reported no difference in the mRNA expression of hormone-sensitive lipase between adipose tissue depots (Montague et al. 1998, Lefebvre et al. 1998).

29 Aims of the study

3 AIMS OF THE STUDY

DHEA and DHEAS are important prohormones. Their metabolism in adipose tissue is of particular interest, since adipose tissue is a major source of biologically active estrogens and androgens in postmenopausal women. In addition to adipose tissue, DHEA may have physiological relevance in another lipophilic environment, i.e., circulating lipoproteins. DHEA fatty acyl esters, naturally occurring lipophilic derivatives of DHEA, are carried in lipoprotein particles in the circulation. Both DHEA and HDL are vascular relaxants. However, the vasodilatory capacity of DHEA fatty acyl esters incorporated in HDL particles has not been previously studied.

The aims of the present study were:

1. To study the fatty acyl esterification of DHEA in human plasma and the association of DHEA fatty acyl esters with human plasma lipoproteins (I)

2. To investigate in isolated rings of rat mesenteric arteries whether HDL-associated DHEA fatty acyl esters improve the vasodilatory capacity of HDL, and to study the role of eNOS, HDL receptor SR-BI as well as androgen and estrogen receptors in the arterial responses (I and II)

3. To study the possible internalization and hydrolysis of HDL- associated DHEA fatty acyl esters in human endothelial cells (II)

4. To study the activity of steroid sulfatase, determined as the conversion of DHEAS to DHEA, and to quantify the expression levels of steroid- converting enzyme genes in abdominal subcutaneous and visceral adipose tissue from pre- and postmenopausal women (III)

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4 MATERIALS AND METHODS

4.1 SUBJECTS AND SAMPLES

The study protocols were approved by the ethics committee of Helsinki University Central Hospital, and written informed consent was obtained from the subjects.

4.1.1 BLOOD SAMPLES FOR INCUBATIONS OF STEROIDS WITH PLASMA Blood samples were obtained from healthy, normolipidemic male and female donors to study the incorporation of radiolabeled DHEA in lipoproteins. Plasma was prepared by immediate centrifugation at 1730 × g, 10 min, +10 °C and used for experiments during the same day (I). For the arterial relaxation studies, blood was drawn from one healthy, normolipidemic, premenopausal woman (44 years of age, normal weight, no medication). Plasma was isolated by centrifugation and used for experiments during the same day (I, II). To examine the internalization and hydrolysis of HDL-associated DHEA fatty acyl esters in cultured endothelial cells, blood was drawn from five healthy, normolipidemic (mean total cholesterol 4.60 mmol/l and triglycerides 1.36 mmol/l) premenopausal women receiving no hormonal medication. Plasma was isolated, pooled, and stored at −80 °C for further experiments (II). To study the phosphorylation of eNOS in cultured endothelial cells, blood samples were taken from five healthy, normolipidemic (mean total cholesterol 4.48 mmol/l and triglycerides 0.98 mmol/l) premenopausal women receiving no hormonal medication. Plasma was isolated, pooled, and stored at −80 °C for further experiments (unpublished).

4.1.2 UMBILICAL CORDS FOR ENDOTHELIAL CELL CULTURES Umbilical cords were obtained after deliveries of term (gestational week 37−42) pregnancies of healthy, nonsmoking women. Endothelial cells were isolated from umbilical veins, cultured, and used freshly for experiments (unpublished).

31 Materials and methods

4.1.3 BLOOD AND ADIPOSE TISSUE SAMPLES FOR ENZYME ACTIVITY, mRNA EXPRESSION, AND HORMONE ASSAYS In Study III, blood and adipose tissue samples were obtained from 18 premenopausal and seven postmenopausal women undergoing elective gynecological surgery for non-malignant reasons. None of the women received systemic estrogen or progestin treatment. Clinical characteristics of the study subjects are shown in Table 1. Blood samples were drawn before the operation. Serum was isolated by centrifugation and stored at −20 °C until the analyses. During the operation, paired abdominal subcutaneous and visceral adipose tissue samples were taken and snap-frozen in liquid nitrogen. The tissue samples were stored at −80 °C until further processing.

Table 1. Clinical characteristics of the patients in Study III.

Premenopausal Postmenopausal n=18 n=7 P valuea Age (years) 43.9 (29.3−51.9) 56.0 (52.3−74.8) <0.001 Body mass index, (kg/m2) 28 (21−35) 25 (22−34) 0.615 Waist-to-hip ratio 0.85 (0.73−0.99) 0.90 (0.82−1.05) 0.130 Serum FSH (IU/l)b 7 (1−18) 70 (26−188) <0.001 Serum SHBG (nmol/l) 61 (31−96) 60 (24−97) 0.929 The data are expressed as median (range). aComparison premenopausal vs. postmenopausal (Mann-Whitney U Test). bReference value for postmenopausal women 26−135 IU/l. FSH, follicle-stimulating hormone; SHBG, sex hormone-binding globulin.

4.2 METHODS

4.2.1 INCUBATION OF STEROIDS WITH PLASMA

4.2.1.1 Incorporation of DHEA in lipoproteins (Study I) Radiolabeled DHEA ([1,2,6,7-3H(N)]DHEA) was added in 0.5 mol/l HEPES buffer (pH 7.4) (ethanol/HEPES 1:4, v/v) to freshly isolated plasma to reach a final concentration of 53 nmol/l (7 × 106 dpm/ml). The incubation was performed at +37 °C for 4 h in the presence and absence of LCAT inhibitor dithionitrobenzoic acid (DTNB; 1.5 mmol/l, final concentration).

32

4.2.1.2 Plasma incubation for cell culture experiments (Study II) To prepare [3H]DHEA fatty acyl ester-enriched HDL, [3H]DHEA in ethanol was added to thawed plasma to reach a final concentration of 212 nmol/l (4.44 × 107 dpm/ml), and the mixture was incubated at +37 °C for 21 h.

4.2.1.3 Plasma incubation for arterial relaxation study (Studies I and II) To prepare steroid fatty acyl ester-enriched HDL, DHEA or estradiol was dissolved in ethanol, diluted with HEPES (ethanol/HEPES 1:4, v/v) and added to freshly isolated plasma to reach a final concentration of 10 μmol/l. The mixture was incubated at +37 °C for 4 h. Plasma incubation with the vehicle (ethanol/HEPES) served as the control. Incubation of DHEA with plasma was also performed in the presence of LCAT inhibitor DTNB (1.5 mmol/l).

4.2.1.4 Plasma incubation for endothelial nitric oxide synthase assay (unpublished) To prepare DHEA fatty acyl ester-enriched HDL, DHEA (Sigma, St. Louis, MO, USA) was dissolved in ethanol, diluted with HEPES buffer (ethanol/HEPES 1:4, v/v) and added to thawed, pooled female plasma to reach a final concentration of 10 μmol/l, and the mixture was incubated at +37 °C for 21 h. Incubation of the vehicle with plasma served as control.

4.2.2 ISOLATION AND PURIFICATION OF LIPOPROTEINS After the incubations, lipoproteins were isolated from plasma by sequential ultracentrifugation (Havel et al. 1955). The density ranges were as follows: VLDL, <1.006 g/ml; LDL, 1.006–1.063 g/ml; HDL, 1.063–1.21 g/ml. Ultracentrifugally isolated lipoproteins were further purified from weakly associated small-molecular-weight substances by size-exclusion chromatography on Sephadex G25 column (column dimension 1 cm × 20 cm). Phosphate-buffered saline (PBS; pH 7.4) was used as elution buffer. Protein concentration in the purified lipoprotein fractions was determined by the method of Lowry et al. (1951). Following purification of lipoproteins containing [3H]-label, radioactivity was determined by liquid scintillation counting.

4.2.3 THIN-LAYER CHROMATOGRAPHY The radioactivity associated with HDL and LDL was further analyzed by thin-layer chromatography (TLC) in Studies I and II. The purified HDL or LDL fraction was extracted four times with ethyl acetate/diethyl ether (1:1,

33 Materials and methods v/v) (3 × sample volume). The organic phase was evaporated, and the extracted lipids were dissolved in methanol and applied to TLC plate (20 cm × 20 cm, Silica gel 60). In Study I, part of the extracted sample was saponified in order to hydrolyze the DHEA fatty acyl esters formed. After saponification in methanolic potassium hydroxide at +60 °C for 2 h, the samples were neutralized with distilled water and HCl, extracted twice with diethyl ether, and applied to TLC. Nonradioactive DHEA and DHEA-3-oleate served as standards. DHEA-3- oleate was synthesized as described in Study I. Hexane/ethyl acetate (7:3, v/v or 1:1, v/v) was used to develop the TLC plate. The locations of the standards were visualized under UV light after rhodamine staining. The locations of the samples containing 3H-label were determined by dividing the lane into 1-cm strips, which were scraped and used for radioactivity measurement.

4.2.4 ARTERIAL RESPONSES IN VITRO (STUDIES I AND II) Relaxation responses to DHEA, estradiol, and different HDLs were studied in isolated rings of rat mesenteric arteries. The mesenteric arteries of male Wistar rats were excised, and endothelium-intact sections (2−3 mm) of arteries were placed between stainless-steel hooks in an organ bath chamber in physiological salt solution. The force of contraction was measured with an isometric force-displacement transducer. Arterial rings were equilibrated for 60 min with the resting tension of 1.5 g, followed by precontraction with 1 μmol/l noradrenaline. The relaxation responses were observed after adding increasing doses of the following components at 6-min intervals: (a) DHEA in ethanol; (b) estradiol in ethanol; (c) HDL isolated after the incubation of DHEA with plasma; (d) HDL from the incubation of DHEA with plasma in the presence of LCAT inhibitor DTNB; (e) HDL from the incubation of estradiol with plasma; (f) native HDL from the plasma incubation with the vehicle (ethanol/HEPES). In selected experiments, the rings were pretreated with the NOS antagonist nitro-L-arginine methyl ester (L-NAME; 0.1 mmol/l, final concentration). In other experiments, the function of HDL receptor SR-BI was attenuated by pretreatment of the arterial rings with thiosemicarbazone BLT-1 (blocker of lipid transport; 1 μmol/l). Furthermore, selected experiments were performed after pretreatment of the rings with estrogen receptor α and β antagonist ICI 182,780 (1 μmol/l) or androgen receptor antagonist bicalutamide (1 μmol/l).

34

4.2.5 ENDOTHELIAL NITRIC OXIDE SYNTHASE ASSAY (UNPUBLISHED)

4.2.5.1 Cell culture Human umbilical vein endothelial cells (HUVEC) were isolated from freshly obtained human umbilical cord veins as described by Jaffe et al. (1973) with some modifications (Ristimäki et al. 1991, Mikkola et al. 1993). Briefly, the cord veins were cannulated and washed three times with 20 ml of phosphate- buffered saline (PBS, pH 7.4; Lonza, Verviers, Belgium), after which the cells were digested with 0.3 g/l collagenase type IV (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 10 min at +37 °C. After collecting the collagenase, the cord veins were washed with medium (Media 199, Life Technologies, Paisley, UK) supplemented with 2 mmol/l L-glutamine (Life Technologies) and 50 mg/l gentamicin (Life Technologies), and the wash medium was added to the collagenase. The cells were then collected by centrifugation (107 × g, 8 min) and resuspended in 5 ml culture medium containing Media 199 supplemented with 2 mmol/l L-glutamine, 50 mg/l gentamicin, 10% fetal bovine serum (HyClone Laboratories, Logan, UT, USA), 3.8 kU/l heparin (Sigma-Aldrich), and 25 mg/l Endothelial Cell Growth Supplement (ECGS; BD Biosciences, Bedford, MA, USA). The cells and the medium were transferred into cell culture flasks, and after maintaining the culture for 2 h at +37 °C, the medium was changed. Thereafter, the medium was changed every second or third day. Confluent cultures were detached with 0.05% Trypsin-EDTA (Life Technologies) and passed at a split ratio of 1:3. The last seeding for the experiment was performed in 75-cm2 flasks in the passage 2−4. Before the experiment, the cells were first preincubated in the culture medium containing phenol red-free Minimum Essential Medium (MEM; Life Technologies), 10% Charcoal/Dextran Treated Fetal Bovine Serum (DCC- FBS; HyClone Laboratories), L-glutamine, and gentamicin for 24 h, and then for additional 12 h in similar medium containing only 2% DCC-FBS. The experimental medium was phenol red-free MEM supplemented with 0.1% BSA (Sigma-Aldrich), L-glutamine, and gentamicin. All culture flasks were pretreated with 0.2% gelatin (Merck Millipore, Billerica, MA, USA) for 30 min at +37 °C.

4.2.5.2 Experimental design DHEA fatty acyl ester-enriched HDL (DHEA ester-enriched HDL) and native HDL were prepared as described in the sections 4.2.1.4 and 4.2.2. HUVECs were treated with DHEA ester-enriched HDL, native HDL, or PBS (vehicle control) for 5, 10, 60, and 180 min. The final HDL protein concentration in the experiment medium was 100 μg/ml. To study the effect of unesterified DHEA without HDL, DHEA (100 nmol/l, final concentration) in ethanol was

35 Materials and methods added to HUVEC culture and incubated for 60 min. Incubation with ethanol (0.02%, v/v) served as control. After the incubations, the media were removed, and the cells were washed twice with PBS and lysed with RIPA buffer (Sigma-Aldrich) containing protease inhibitor cocktail (Sigma- Aldrich). In every experiment, one flask of cells was lysed before any treatment at the baseline. The cell lysates were stored at −140 °C. Total protein concentrations of the cell lysates were determined by Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA).

4.2.5.3 Western blot analysis Western blot analysis was carried out to examine protein levels of eNOS and serine-1177-phosphorylated eNOS in HUVECs after the treatment with DHEA ester-enriched HDL, native HDL, PBS control, DHEA, or ethanol control. Western blot analysis was performed as described (Rahkola-Soisalo et al. 2013) with some modifications. Of the HUVEC lysates, an aliquot of 3.75 μg of protein was used for gel electrophoresis. Primary antibodies were mouse monoclonal anti-eNOS/NOS type III (1:2500; BD Biosciences) and anti-eNOS (pS1177) (1:750; BD Biosciences). As a secondary antibody, polyclonal rabbit anti-mouse immunoglobulin coupled to horseradish peroxidase (DAKO, Glostrup, Denmark) was used. Glyceraldehyde-3- phosphate dehydrogenase (GAPDH, 1:20000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) served as a protein-loading control. Signal intensity was quantified by densitometry (Syngene; Synoptics Limited, Cambridge, UK). The results were normalized to the signal intensity of the baseline sample on the same membrane. We performed two independent cell culture experiments, and three Western blot analyses from each experiment.

4.2.6 CELLULAR UPTAKE AND HYDROLYSIS OF HDL-ASSOCIATED DHEA FATTY ACYL ESTERS (STUDY II) HUVECs were purchased from Lonza Group Ltd. (USA) and cultured as described in Study II. To examine the uptake of HDL-associated [3H]DHEA esters, cells in 6-well plates were treated with [3H]DHEA fatty acyl ester- enriched HDL in serum-free medium for 0.5, 1, 3, 6, or 24 h. At each time point, one well containing no cells served as control. In selected experiments, the function of HDL receptor SR-BI was blocked by pretreating the cells or media (in control wells) with thiosemicarbazone BLT-1. After the incubation, the cell culture media and the control media were collected and centrifuged. The cells were washed twice with PBS, and intracellular lipids were extracted by treating the cell monolayer with hexane/isopropanol (2:3, v/v) for 1 h at room temperature. The intracellular lipid extract was evaporated and stored in methanol. The cell remnants were lysed with sodium hydroxide, and the protein concentration was measured.

36

To study the metabolism of DHEA fatty acyl esters in HUVECs, the cells and media (in control wells) were incubated with [3H]DHEA fatty acyl ester- enriched HDL for 6 h, after which the media were removed, and the cells were washed twice with PBS. After changing fresh culture media without [3H]DHEA ester-HDL, the cells were cultured for additional 12 h or 36 h. The cells and media were then collected and processed as described above. The radioactivity in media and cellular fractions was measured by liquid scintillation counting. The [3H]-labeled steroids were extracted from the media with ethyl acetate/diethyl ether (1:1, v/v), evaporated and dissolved in methanol. The distribution of [3H]DHEA and [3H]DHEA fatty acyl esters in the cells and the media was analyzed by TLC as described in the section 4.2.3.

4.2.7 STEROID SULFATASE ACTIVITY ASSAY IN ADIPOSE TISSUE (STUDY III) Radiolabeled DHEAS was used as a substrate to study the activity of steroid sulfatase in adipose tissue. Paired subcutaneous and visceral adipose tissue samples (200 mg each) were homogenized in distilled water on ice. Purified [3H]DHEAS (Fotsis 1987) in ethanol was added to the tissue homogenate, and the mixture was incubated at +37 °C for 3 h. To observe the hydrolysis of [3H]DHEAS as a function of time, [3H]DHEAS was incubated with pooled subcutaneous and visceral adipose tissue homogenate for 0, 1, 2, 3, 6, and 24 h at +37 °C. Incubations of [3H]DHEAS with water served as controls. After the incubation period, the mixture was extracted with diethyl ether, evaporated, dissolved in 1 ml of hexane, and subjected to Sephadex LH-20 chromatography (column dimension 1 × 6 cm) to purify [3H]DHEA formed during the incubation. The hydrophobic fraction was eluted with 8 ml of hexane, after which the column was washed with 6 ml of hexane, and the DHEA fraction was eluted with 17 ml of chloroform/hexane (1:1, v/v). The radioactivity in the DHEA fraction was measured by liquid scintillation counting.

4.2.8 QUANTITATIVE REAL-TIME RT-PCR (STUDY III) The mRNA expression levels of six steroid-metabolizing enzymes, namely steroid sulfatase (STS), hormone-sensitive lipase (LIPE), aromatase (CYP19A1), and 17βHSD types 1, 7, and 12 (HSD17B1, HSD17B7, HSD17B12) in adipose tissue were quantified by real-time RT-PCR as previously described (Wang F et al. 2013). Briefly, frozen adipose tissue samples were homogenized in Trizol, and total RNA was isolated and purified. RNA was reverse transcribed into cDNA, and mRNA expression levels of the six target genes were analyzed by quantitative real-time RT-PCR. The efficiency ranged between 93% and 114%. The results were normalized to the geometric mean

37 Materials and methods of the two most stable control genes, importin 8 (IPO8) and lysine (K)- specific demethylase 2B (KDM2B).

4.2.9 QUANTIFICATION OF SERUM HORMONES (STUDY III) Quantification methods for serum DHEAS, DHEA, estrone, and estradiol concentrations by liquid chromatography−tandem mass spectrometry are described in the original Study III.

4.2.10 OTHER METHODS Serum follicle-stimulating hormone (FSH) and SHBG were measured with commercial methods described in Study III.

4.2.11 STATISTICAL ANALYSES Statistical analyses were carried out with SPSS software (versions 15.0, 17.0, and 22.0). Data from arterial relaxation experiments (Studies I and II) are presented as mean±standard error of the mean (SEM). Differences in mean relaxation responses were tested by ANOVA followed by Sheffe’s post hoc test, or by independent-samples t-test when comparing two groups. In Study III, the data are expressed as median (range or interquartile range). The Mann-Whitney U test was performed for between-group differences, and the Wilcoxon signed rank test for pairwise comparisons. Correlation analyses were performed using Spearman’s nonparametric correlation. The level of significance was P<0.05. When multiple correlation analyses between hormone concentrations and gene expression levels were carried out, the correlations were considered significant at P<0.01. The unpublished data are presented as mean±SEM, and the differences were tested by ANOVA followed by Sheffe’s post hoc test, or by independent-samples t-test.

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5 RESULTS

5.1 FORMATION OF DHEA FATTY ACYL ESTERS IN HUMAN PLASMA (STUDY I)

To study whether DHEA becomes incorporated in lipoproteins, [3H]DHEA was incubated with human plasma, after which lipoproteins were isolated and purified. Of the radioactivity recovered in the lipoprotein fractions, 2% was in VLDL fraction, 39% in LDL, and 59% in HDL. The radioactivity coincided with the protein in purified lipoprotein fractions, indicating that [3H]DHEA was associated with lipoproteins (Figure 3). When the plasma incubation was carried out in the presence of LCAT inhibitor DTNB, almost no radioactivity was recovered in the lipoprotein fractions, suggesting that fatty acyl esterification via LCAT function is required for DHEA to become associated with lipoproteins (Figure 3). After the purification with Sephadex G25, the lipoprotein-containing fractions from the incubation without DTNB were further analyzed by TLC. Part of each sample was saponified in order to hydrolyze DHEA fatty acyl esters formed during the incubation. Of the radioactivity in the nonhydrolyzed HDL and LDL samples, 95% and 92%, respectively, recovered in the same position with the DHEA fatty acyl ester standard, confirming that DHEA was associated with lipoproteins in esterified form. The hydrolyzed samples comigrated with the DHEA standard.

A HDL B LDL 7 7 [3H]DHEA 6 6 [3H]DHEA + LCAT inhibitor dpm) dpm) 5 5 5 5 4 4 3 3 2 2 1 1 Radioactivity (x10 Radioactivity (x10 0 0 0246810 0246810 Fraction No. Fraction No.

Figure 3. Radioactivity elution pattern of plasma HDL (A) and LDL (B) after chromatography on Sephadex G25. [3H]DHEA was incubated with plasma for 4 h in the presence (○) and absence (●) of LCAT inhibitor DTNB, followed by isolation and purification of lipoproteins. Arrows indicate the fractions where HDL and LDL particles elute. Study I.

39 Results

5.2 ARTERIAL RESPONSES IN VITRO (STUDIES I AND II)

5.2.1 ARTERIAL RESPONSES TO DHEA AND ESTRADIOL DHEA and estradiol caused a dose-dependent relaxation in isolated rings of rat mesenteric arteries. The relaxation response to DHEA was stronger than the response to estradiol at the concentration of 100 nmol/l or higher (Figure 4).

0 DHEA E 20 ** 2 40 ** 60 * 80

Arterial relaxation100 % **

-9 -8 -7 -6 -5 -4 Concentration / log (M)

Figure 4. Relaxation responses to DHEA and estradiol (E2) in isolated rat arterial rings. Endothelium-intact rings were precontracted with noradrenaline, and the relaxation responses were measured after adding increasing doses (1 nmol/l–100 μmol/l) of DHEA (n=11) or E2 (n=10). The data are expressed as mean±SEM. *P<0.05, **P<0.01, comparison DHEA vs. E2 (Independent-samples t-test). Study I.

5.2.2 ARTERIAL RESPONSES TO STEROID FATTY ACYL ESTER- ENRICHED HDL AND NATIVE HDL To explore the possibility that DHEA fatty acyl esters contained in HDL could modify the vasodilatory effect of HDL, precontracted rat arterial rings were treated with increasing doses of HDL obtained after the incubation of DHEA with plasma. This DHEA fatty acyl ester-enriched HDL caused a dose- dependent relaxation, which was significantly stronger than the relaxation response to native HDL obtained from the plasma incubation with the vehicle (maximal relaxation responses 43% vs. 25%, P<0.001) (Figure 5A). HDL isolated after the incubation of DHEA with plasma in the presence of LCAT inhibitor DTNB caused a dose-dependent relaxation (maximal 25%), which was weaker than the response to DHEA fatty acyl ester-enriched HDL (P<0.001) and did not differ from the response to native HDL (P=0.98) (Figure 5A). Experiments with HDL enriched with fatty acyl esterified estradiol were also carried out as this steroid is generally regarded as a potent vasodilator. HDL obtained after the incubation of estradiol with plasma caused a dose- dependent relaxation, which was significantly stronger than the response to

40

native HDL at the HDL total protein concentrations of 1, 10, and 40 μg/ml, but not at the maximal concentration of 125 μg/ml (maximal relaxation responses 33% vs. 25%, P=0.062) (Figure 5B).

Native HDL A B Native HDL DHEA-FAE-HDL E2-FAE-HDL HDL (DHEA+DTNB incubation with plasma) 0 0

10 ** 10 * ** 20 20 * ** 30 30 * ** 40 40 Arterial relaxation % ** Arterial relaxation % 50 50 0.1 1 10 100 1000 0.1 1 10 100 1000 HDL total protein (μg/ml) HDL total protein (μg/ml)

Figure 5. Relaxation responses to HDL in isolated endothelium-intact rat mesenteric arterial rings. The rings were precontracted with noradrenaline, and the relaxation responses to increasing doses of HDL (0.1–125 μg/ml) were measured. (A) Relaxation response to HDL isolated after plasma incubation with 10 μmol/l DHEA (DHEA fatty acyl ester-enriched HDL, DHEA-FAE-HDL), with 10 μmol/l DHEA + LCAT inhibitor DTNB, or with the vehicle (Native HDL). (B) Relaxation response to HDL isolated after plasma incubation with 10 μmol/l estradiol (E2-FAE-HDL) or with the vehicle (Native HDL). The data are expressed as mean±SEM, n=13–24. *P<0.05, **P<0.01, compared to native HDL. Study I.

5.2.3 EFFECT OF BLOCKING NITRIC OXIDE SYNTHASE AND FUNCTION OF HDL, ANDROGEN, AND ESTROGEN RECEPTORS To examine the mechanism by which DHEA fatty acyl ester-enriched HDL causes vasorelaxation, the arterial rings were pretreated with the NOS antagonist L-NAME. After the pretreatment, the relaxation response to DHEA ester-enriched HDL was significantly weaker than the response without pretreatment (maximal 43% vs. 30%, P=0.008) (Figure 6A), indicating that the vascular relaxation response was partly mediated by eNOS. Pretreatment with L-NAME impaired the relaxation response to estradiol ester-enriched HDL, as well (33% vs. 18%, P=0.014) (Figure 6B). The response to native HDL was slightly impaired by pretreatment with L-NAME (25% vs. 19%), but the difference failed to reach statistical significance (P=0.062). To study the role of HDL receptor SR-BI in the vasorelaxation induced by HDL-associated DHEA fatty acyl esters, the rat arterial rings were pretreated with SR-BI inhibitor, BLT-1. This pretreatment markedly reduced the relaxation response to DHEA ester-enriched HDL (maximal relaxation 74% vs. 18%, P<0.001) (Figure 6C), suggesting that the vasodilation was in major part mediated by the SR-BI receptor pathway. Pretreatment with

41 Results

E -FAE-HDL A DHEA-FAE-HDL B 2 NOS antagonist + DHEA-FAE-HDL NOS antagonist + E2-FAE-HDL 0 0

10 10

20 20 * 30 30 *

40 40 * Arterial relaxation % Arterial relaxation % ** 50 50 0.1 1 10 100 1000 0.1 1 10 100 1000 HDL total protein (μg/ml) HDL total protein (μg/ml)

C DHEA-FAE-HDL D Native HDL SR-BI blocker + DHEA-FAE-HDL SR-BI blocker + Native HDL 0 0 10 10 20 20 30 ** 30 ** 40 40 50 50 ** 60 ** 60 ** 70 ** 70 ** Arterial relaxation % Arterial relaxation 80% 80 90 ** 90 1101001000 1101001000 HDL total protein (μg/ml) HDL total protein (μg/ml)

DHEA-FAE-HDL E AR blocker + DHEA-FAE-HDL ER blocker + DHEA-FAE-HDL 0 10 20 30 40 50 60 70

Arterial relaxation 80% 90 1101001000 HDL total protein (μg/ml)

Figure 6. Relaxation responses to HDL in isolated rat mesenteric arterial rings. The rings were precontracted with noradrenaline, after which the relaxation responses to increasing doses of HDL (0.1–125 μg/ml or 1–125 μg/ml) were measured. (A) and (B) Relaxation responses to DHEA fatty acyl ester-enriched HDL (DHEA-FAE-HDL) and estradiol fatty acyl ester-enriched HDL (E2-FAE-HDL) without pretreatment and after pretreatment with NOS antagonist L-NAME. (C) and (D) Responses to DHEA- FAE-HDL and native HDL with and without pretreatment with SR-BI inhibitor. (E) Response to DHEA-FAE-HDL with and without pretreatment with androgen (AR) and estrogen (ER) receptor inhibitors. Data are expressed as mean±SEM. *P<0.05, **P<0.01, pretreated vs. without pretreatment. Studies I and II.

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BLT-1 also impaired the relaxation response to native HDL (56% vs. 14%, P=0.001) (Figure 6D). We further studied the possibility that vasodilation caused by DHEA fatty acyl ester-enriched HDL could be mediated through conversion of DHEA to androgens or estrogens, and thus through androgen or estrogen receptors. Pretreatment with bicalutamide, an androgen receptor antagonist, did not affect the vasorelaxation response to DHEA ester-enriched HDL (Figure 6E). Blocking of estrogen receptors α and β by preincubation with ICI 182,780, which is also an agonist of G protein-coupled estrogen receptor (Meyer et al. 2009), enhanced the vasorelaxation response to DHEA ester- enriched HDL slightly but nonsignificantly (Figure 6E).

5.3 PHOSPHORYLATION OF ENDOTHELIAL NITRIC OXIDE SYNTHASE IN ENDOTHELIAL CELLS (UNPUBLISHED)

To further explore the mechanisms by which DHEA fatty acyl ester-enriched HDL causes arterial relaxation, we studied in cultured endothelial cells whether these HDL particles increase the phosphorylation degree of eNOS. Treatment of the cells with DHEA ester-enriched HDL increased serine-1177- phosphorylated eNOS at the maximum 9.2-fold compared to the PBS control (Figures 7 and 8A). However, this effect did not significantly differ from the eNOS phosphorylation caused by native HDL (9.0-fold compared to the control). A 60-min treatment with free DHEA without HDL caused a 2.2-fold increase in phosphorylated eNOS compared to the ethanol control (Figure 8A). Total eNOS signal remained unchanged (Figures 7 and 8B) (Paatela H et al. unpublished results).

Figure 7. A representative Western blot analysis of eNOS in endothelial cells. HUVECs were treated with native HDL, DHEA fatty acyl ester-enriched HDL (DHEA-FAE-HDL), or PBS (control). HDL total protein concentration during incubation was 100 µg/ml. Cell lysates were analyzed by Western blot using monoclonal antibodies against serine-1177-phosphorylated eNOS (p-eNOS) and total eNOS.

43 Results

A 12 Native HDL 10 * * DHEA-FAE-HDL PBS control 8 DHEA 100 nmol/l 6 * * Ethanol control 4 # 2 p-eNOS (fold control) 0 51060180 B 2

1

eNOS (fold control) (fold eNOS 0 51060180 Incubation time (min)

Figure 8. Protein levels of eNOS in HUVECs. (A) Serine-1177-phosphorylated eNOS (p-eNOS). (B) Total eNOS. HUVECs were treated with native HDL, DHEA fatty acyl ester-enriched HDL (DHEA-FAE-HDL), or PBS (control). To study the effect of free DHEA, cells were treated with 100 nmol/l DHEA or ethanol (control) for 60 min. Cell lysates were analyzed by Western blot, followed by densitometric analysis. The NOS scales represent density in arbitrary units relative to PBS or ethanol control. The data are expressed as mean+SEM, n=5–6. *P≤0.01 compared to PBS control (ANOVA and Sheffe’s post hoc test), #P=0.014 compared to ethanol control (Independent-samples t-test).

5.4 CELLULAR UPTAKE AND HYDROLYSIS OF HDL- ASSOCIATED DHEA FATTY ACYL ESTERS (STUDY II)

We further explored whether the vasodilatory effect of DHEA ester-enriched HDL could be mediated by rapid cellular uptake and hydrolysis of DHEA fatty acyl esters transported by HDL. This possible mechanism was studied in cultured human endothelial cells. HDL enriched with [3H]DHEA fatty acyl esters was prepared by incubating [3H]DHEA with pooled female plasma, followed by isolation and purification of HDL. HUVECs were treated with this [3H]DHEA ester-enriched HDL, and after the incubation period, intracellular and extracellular radioactivity was analyzed. Intracellular radioactivity increased as a function of time to the maximum of 1.3% of total radioactivity (Figure 9A). To investigate whether DHEA fatty acyl esters are hydrolyzed after the internalization, the intracellular and extracellular radioactivity was further analyzed by TLC. Intracellularly, the proportion of free, unesterified [3H]DHEA was relatively stable up to 6 h, but then

44

AB

1.5 4.0 Cells 3.5 Cell culture medium 3.0 Control medium 1.0 2.5 2.0 1.5 0.5 1.0

radioactivity on TLC on radioactivity 0.5 H]DHEA, % of recovered recovered of % H]DHEA, 3 [

Intracellular radioactivity0.0 (%) 0.0 024681012141618202224 0.5 h 1 h 3 h 6 h 24 h Incubation time (h) Incubation time

Figure 9. Uptake and hydrolysis of HDL-associated [3H]DHEA fatty acyl esters in HUVECs. (A) Uptake of HDL-associated [3H]DHEA by HUVECs as a function of time. Cells were incubated with [3H]DHEA fatty acyl ester-enriched HDL (HDL protein 100 μg/ml), and intracellular radioactivity was measured. Intracellular radioactivity is expressed as a percentage of the total radioactivity in cells and medium (mean±SEM). (B) Radioactivity in unesterified DHEA fraction in TLC analysis. Cells or control media without cells were incubated with [3H]DHEA ester-enriched HDL, after which media and cells were extracted, and subjected to TLC. Data are expressed as a percentage radioactivity in unesterified DHEA fraction of the total radioactivity recovered. Study II. increased from 1.6% up to 3.4% between 6- and 24-h incubations (Figure 9B). Similarly to what was observed in the intracellular compartment, in the cell culture media, the proportion of free [3H]DHEA increased between 6 and 24 h from 1.2% to 3.4% (Figure 9B). In the control wells, containing media and [3H]DHEA ester-enriched HDL but no cells, the proportion of free [3H]DHEA remained relatively stable, indicating that [3H]DHEA esters were not spontaneously hydrolyzed (Figure 9B). We next studied the role of SR-BI in the internalization of HDL- associated DHEA fatty acyl esters. Treatment of the endothelial cells with SR-BI inhibitor BLT-1 prior to exposure to [3H]DHEA fatty acyl ester- enriched HDL inhibited cellular uptake of [3H]DHEA esters by 20–38% (Figure 10A). After 24-h incubation, the proportion of unesterified DHEA in the cell culture medium, analyzed by TLC, was 7.2% of the recovered radioactivity, but decreased to 2.1% when the cells were pretreated with BLT-1 (Figure 10B). Thus, this pretreatment inhibited the secretion of free DHEA, after the intracellular hydrolysis of DHEA esters, from the cells more than the uptake of DHEA esters. We further studied the metabolism of DHEA fatty acyl esters after the uptake by HUVECs. Cells were incubated with [3H]DHEA ester-enriched HDL for 6 h, after which the media were changed and the cells were cultured for additional 12 and 36 h. At these time points, the distribution of [3H] was analyzed by TLC. In the cells, the proportion of [3H]DHEA fatty acyl esters decreased and free [3H]DHEA markedly increased from 12 to 36 h

45 Results

(Figure 11). In the cell culture media, at these time points, the radioactivity, representing secretion of [3H]-labeled steroids by the cells, recovered mostly in the free, unesterified steroid fraction (Figure 11). Part of the radioactivity in the media was recovered near the sample application point on the TLC plate, suggesting that the cells had secreted unidentified hydrophilic metabolites of [3H]DHEA.

ABCell culture medium, BLT-1 - Control medium, BLT-1 - BLT-1 - Cell culture medium, BLT-1+ 1.0 BLT-1 + 8 Control medium, BLT-1 + 7 0.8 6

0.6 5 4 0.4 3 2 0.2 radioactivity on TLC on radioactivity 1 H]DHEA, % of recovered recovered of % H]DHEA, 3 [

Intracellular radioactivity0.0 (%) 0 024681012141618202224 0.5 h 1 h 3 h 24 h Incubation time (h) Incubation time

Figure 10. Effect of blocking SR-BI function on uptake and hydrolysis of HDL-associated [3H]DHEA fatty acyl esters in HUVECs. (A) Uptake of HDL-associated [3H]DHEA esters as a function of time. Cells were pretreated with SR-BI inhibitor BLT-1 prior to incubation with [3H]DHEA ester-enriched HDL (HDL protein 100 μg/ml). After the incubation, intracellular radioactivity was measured. Intracellular radioactivity is expressed as a percentage of the total radioactivity in cells and medium. (B) Cells or control media without cells were pretreated with BLT-1 and then incubated with [3H]DHEA ester-enriched HDL. After the incubation, media were extracted and subjected to TLC. Data are expressed as a percentage radioactivity in unesterified DHEA fraction of the total radioactivity recovered. Study II.

100 DHEA fatty acyl ester Free DHEA 80 Polar metabolites of DHEA

60

40 Mediumchanged

20 % of recovered radioactivity recovered of % 0 6 h 12 h 12 h 36 h 36 h medium medium cells medium cells

Figure 11. Radioactivity as percentage distribution in HUVECs and culture media. HUVECs were incubated with [3H]DHEA fatty acyl ester-enriched HDL for 6 h. After changing fresh medium without [3H]DHEA ester-HDL, the cells were incubated for additional 12 or 36 h, and media and cells were extracted and subjected to TLC. Study II.

46

5.5 ACTIVITY OF STEROID SULFATASE AND GENE EXPRESSION OF STEROID-CONVERTING ENZYMES IN ADIPOSE TISSUE (STUDY III)

5.5.1 SERUM HORMONE CONCENTRATIONS Concentrations of DHEAS, DHEA, estrone, and estradiol in serum obtained from 18 premenopausal and seven postmenopausal women undergoing elective surgery are shown in Table 2. All these hormone concentration were higher in premenopausal than in postmenopausal women, and the concentrations correlated negatively with age (DHEAS, r=−0.581; DHEA, r=−0.719; estrone, r=−0.513; estradiol, r=−0.467; P<0.05 for all; n=25). Serum DHEA correlated positively with serum DHEAS in premenopausal (r=0.734, P=0.001, n=18), in postmenopausal (r=0.893, P=0.007, n=7), and in all women analyzed together (r=0.843, P<0.001, n=25). Furthermore, serum estradiol correlated with serum estrone in premenopausal (r=0.796, P<0.001), postmenopausal (r=1.0, P<0.001), and all women (r=0.898, P<0.001).

Table 2. Serum hormone concentrations. Study III.

Premenopausal Postmenopausal n=18 n=7 P valuea DHEAS, μmol/l 3.7 (1.2−7.1) 1.5 (0.2−4.2) 0.025 DHEA, nmol/l 14.1 (1.9−58.0) 4.7 (0.2−20.5) 0.012 Estrone, pmol/l 228 (122−970) 94 (15−216) 0.001 Estradiol, pmol/l 339 (33−1342) 42 (9−358) 0.005 The data are expressed as median (range). aComparison premenopausal vs. postmenopausal (Mann-Whitney U Test).

5.5.2 STEROID SULFATASE ACTIVITY IN ADIPOSE TISSUE To investigate steroid sulfatase activity in adipose tissue, radiolabeled DHEAS was used as the substrate, and the conversion of [3H]DHEAS to [3H]DHEA during the incubation in the presence of adipose tissue sample was determined. The hydrolysis of [3H]DHEAS in pooled subcutaneous and visceral adipose tissue homogenate increased as a function of time up to 24 h (Figure 12A). Based on this assay, we chose the 3-h incubation time for further experiments. Next, we incubated [3H]DHEAS in the presence of homogenized subcutaneous or visceral adipose tissue derived from pre- and postmenopausal women, and observed the conversion of [3H]DHEAS to [3H]DHEA. Steroid sulfatase activity was higher in postmenopausal than in premenopausal women both in subcutaneous (median 379 vs. 257 pmol converted to [3H]DHEA/kg adipose tissue per hour; P=0.006) and in visceral

47 Results

(545 vs. 360 pmol/kg/hour; P=0.004) adipose tissue (Figure 12B). In visceral adipose tissue, steroid sulfatase activity was higher compared to the activity in subcutaneous adipose tissue. This difference was significant in premenopausal (P=0.035) and all women (475 vs. 303 pmol/kg/hour, P=0.010), but not in postmenopausal women (P=0.128) (Figure 12B). In premenopausal women and in all women analyzed together, DHEA sulfatase activity in subcutaneous fat correlated positively with the activity in visceral fat (premenopausal, r=0.472, P=0.048; all, r=0.571, P=0.003). Steroid sulfatase activity in both adipose tissue depots correlated positively with age (visceral, r=0.416, P=0.039; subcutaneous, r=0.604, P=0.001) in all women analyzed together. However, when premenopausal and postmenopausal women were analyzed separately, no positive correlation was found between sulfatase activity and age. Steroid sulfatase activity did not correlate with serum DHEAS or DHEA concentrations in any of the study groups.

AB [3H]DHEAS + adipose tissue Premenopausal 50 # # Control 600 Postmenopausal 40 500 * 400 30 * 300 20

Hydrolysis 200

Hydrolysis (%) 10 100 (pmol/kg per hour) per (pmol/kg 0 0 024681012141618202224 Sc Visc Incubation time (h) Adipose tissue depot

Figure 12. Hydrolysis of DHEAS in adipose tissue. (A) Conversion of [3H]DHEAS to [3H]DHEA as a function of time. [3H]DHEAS was incubated in the presence or absence (control) of homogenized, pooled subcutaneous and visceral adipose tissue samples, followed by extraction, purification, and quantification of [3H]DHEA. Data are expressed as a percentage of [3H]DHEAS converted to [3H]DHEA. (B) Steroid sulfatase activity in subcutaneous (Sc) and visceral (Visc) adipose tissue. [3H]DHEAS was incubated with fat tissue homogenate for 3 h, followed by extraction, purification, and quantification of [3H]DHEA. The hydrolysis is expressed as the amount of liberated [3H]DHEA (pmol)/adipose tissue (kg)/incubation time (h). Data are expressed as median (interquartile range). Premenopausal, n=18; Postmenopausal, n=7. #P<0.01, pre- vs. postmenopausal (Mann-Whitney U Test). *P<0.05, subcutaneous vs. visceral fat (Wilcoxon Signed Rank Test). Study III.

5.5.3 GENE EXPRESSION OF STEROID-CONVERTING ENZYMES IN ADIPOSE TISSUE The relative mRNA expression of STS gene in adipose tissue was similar in premenopausal and postmenopausal women (Figure 13). STS displayed, however, higher expression in subcutaneous than in visceral adipose tissue. This difference was significant in premenopausal (P=0.001) and in all

48

Premenopausal Postmenopausal 0.8 * # 0.7 * 0.6 #

0.5 *

0.4 * *

0.3 # * 0.2

Relative mRNAexpression Relative 0.1 * * 0.0 Sc Sc Sc Sc Sc Sc Visc Visc Visc Visc Visc Visc STS LIPE STS LIPE CYP19A1 HSD17B1 HSD17B7 HSD17B12 CYP19A1 HSD17B1 HSD17B7

HSD17B12

Figure 13. Relative mRNA expression levels of steroid-converting genes in abdominal subcutaneous (Sc) and visceral (Visc) adipose tissue derived from pre- and postmenopausal women. STS, steroid sulfatase; LIPE, hormone-sensitive lipase; CYP19A1, aromatase; HSD17B1, HSD17B7, HSD17B12, 17β-hydroxysteroid dehydrogenase types 1, 7, and 12. Data are presented as median (interquartile range). #P≤0.01, pre- vs. postmenopausal (Mann-Whitney U Test). *P<0.01, comparison of the expression of the same gene in subcutaneous vs. visceral fat (Wilcoxon Signed Rank Test). Study III. women (P<0.001), but nonsignificant in postmenopausal women (P=0.080) (Figure 13). In addition to STS gene, we quantified gene expression levels of some steroid-converting enzymes that drive towards the formation of active estradiol. The mRNA expression levels of CYP19A1 and HSD17B12 were higher in postmenopausal compared to premenopausal subcutaneous adipose tissue. Similarly in visceral adipose tissue, the expression of LIPE gene was higher in postmenopausal than in premenopausal women (Figure 13). In comparison between the two adipose tissue depots, the expression levels of LIPE and CYP19A1 were higher in subcutaneous adipose tissue in premenopausal (Figure 13) and all women (LIPE, P=0.004; CYP19A1, P=0.001), and the expression of HSD17B7 was higher in visceral adipose tissue in premenopausal (Figure 13) and all women (P<0.001). We found no measurable mRNA expression of estrogen sulfotransferase gene (SULT1E1) in adipose tissue. The DHEA sulfatase activity correlated positively with the mRNA expression levels of STS, CYP19A1, and HSD17B12 in subcutaneous adipose

49 Results tissue in all women analyzed together (Table 3). In visceral adipose tissue, the sulfatase activity correlated with CYP19A1 gene expression (Table 3). In postmenopausal women, the STS mRNA expression in subcutaneous and visceral adipose tissue correlated strongly with serum estrone and estradiol concentrations (Table 4). Serum DHEAS and DHEA concentrations displayed, however, no correlation with the mRNA expression levels of any of the genes studied. The mRNA expression of CYP19A1 in subcutaneous adipose tissue correlated positively with waist-to-hip ratio in premenopausal (r=0.574, P=0.016) and all women (r=0.541, P=0.008), and the expression of the same gene in visceral fat displayed a positive correlation with body mass index (r=0.886, P=0.019) and waist-to-hip ratio (r=0.829, P=0.042) in postmenopausal women.

Table 3. Correlations between steroid sulfatase activity and mRNA expression levels of steroid-converting genes in adipose tissue. Study III.

STS LIPE CYP19A1 HSD17B1 HSD17B7 HSD17B12 Sulfatase activity Subcutaneous 0.412* 0.045 0.694** -0.013 0.118 0.536** Visceral 0.042 -0.059 0.414* 0.242 0.269 0.319 Correlation coefficients between the corresponding variables are expressed in the table (Spearman’s correlation). All women (n=24) analyzed together. *P<0.05, **P<0.01, Statistically significant correlations are in bold.

Table 4. Correlations between serum estrogen concentrations and adipose tissue mRNA expression of steroid sulfatase (STS) in postmenopausal women. Study III.

Serum hormone Subcutaneous STS mRNA Visceral STS mRNA r P-value r P-value Estrone 0.943 0.005 0.943 0.005 Estradiol 0.943 0.005 0.943 0.005 r, correlation coefficient (Spearman’s correlation). n=6. Statistically significant correlations are in bold.

50

6 DISCUSSION

6.1 INCORPORATION OF DHEA FATTY ACYL ESTERS IN PLASMA LIPOPROTEINS

During the incubation with human plasma, [3H]DHEA became fatty acyl esterified and associated with lipoproteins (I), which is in line with previous studies (Leszczynski et al. 1989, Roy and Bélanger 1993, Lavallée et al. 1996a). The esterification of DHEA occurs in HDL3 subfraction (Leszczynski et al. 1989, Leszczynski and Schafer 1991), and from these particles, lipophilic DHEA fatty acyl esters can be transferred to other lipoprotein particles (Roy and Bélanger 1989, Provost et al. 1997). In our study, after the 4-hour incubation of [3H]DHEA with plasma, [3H]DHEA fatty acyl esters were found in HDL, LDL, and VLDL fractions, suggesting that DHEA esters had been transferred from HDL to other lipoproteins. Consistent with previous studies (Roy and Bélanger 1989, Lavallée et al. 1996a), the esterification was dependent on LCAT. The radioactivity associated with HDL and LDL was almost exclusively (92-95%) in DHEA fatty acyl esterified form, similarly to a previous finding that approximately 90% of DHEA in lipoprotein fractions after 6-hour plasma incubation was in lipophilic form (Roy and Bélanger 1989). In our study, when LCAT inhibitor DTNB was present in the incubation, almost no radioactivity was associated with lipoproteins, indicating that esterification is required for the incorporation of DHEA in the lipoproteins. DHEA becomes esterified in plasma at much higher rate than estradiol (Jones and James 1985). In comparison with a previous study by our group (Helisten et al. 2001), determining esterification of estradiol during 24-hour plasma incubation, we now observed in HDL fraction approximately four times higher amounts of esterified DHEA compared to esterified estradiol in the previous study, the amounts of DHEA and estradiol added to the plasma being equal.

6.2 VASODILATORY EFFECTS OF DHEA AND ESTRADIOL

On the vascular endothelium, both DHEA and estradiol rapidly activate eNOS, thus resulting in the production of nitric oxide (Simoncini et al. 2003, Chambliss and Shaul 2002), a potent vasodilator. We aimed to confirm the rapid vasodilatory effects of DHEA and estradiol in our experimental system in isolated rings of rat mesenteric arteries and to compare the relaxation responses to these two steroids. Both DHEA and estradiol caused a dose- dependent relaxation in the arterial rings (I). DHEA induced a stronger vasodilation compared to estradiol. Both steroids caused the arterial

51 Discussion relaxation at rather high concentrations. The lowest concentration of estradiol causing a significant (20%) relaxation was 10 μmol/l, which is a clearly supraphysiological concentration. With DHEA, approximately similar relaxation response (24%) was achieved at the concentration of 1 μmol/l, also a supraphysiological concentration (Labrie et al. 2011). Based on these relaxation responses ex vivo and the fact that DHEA is circulating at higher concentrations than estradiol, our present findings suggest that circulating DHEA could be a more efficient vasodilator compared to estradiol.

6.3 VASCULAR EFFECTS OF HDL-ASSOCIATED STEROID FATTY ACYL ESTERS

6.3.1 ENHANCEMENT OF VASODILATORY EFFECTS OF HDL Both HDL particles and DHEA have been shown to increase endothelial nitric oxide production and cause rapid vasodilation (Yuhanna et al. 2001, Liu and Dillon 2002, Simoncini et al. 2003, Liu and Dillon 2004). This led us to ask whether HDL-associated DHEA fatty acyl esters could improve the vasodilatory effect of HDL. In isolated rings of rat mesenteric arteries, we demonstrated that HDL enriched with DHEA fatty acyl esters was a more potent vasodilator than native HDL (I). DHEA ester-enriched HDL was prepared by incubating DHEA with plasma. When identical plasma incubation was performed in the presence of LCAT inhibitor, HDL isolated after the incubation caused significantly weaker vasodilation compared to DHEA fatty acyl ester-enriched HDL, and the relaxation response did not differ from the response to native HDL (I). This finding indicates that the esterification of DHEA is required for the improvement of the vasodilatory response. Steroid fatty acyl esters are lipophilic and thus incorporated in the lipoprotein particles in the circulation (Roy and Bélanger 1993, Vihma et al. 2003a). The biological role of lipoprotein-associated steroid fatty acyl esters is not fully understood. HDL has several antiatherogenic properties, which are thought to be dependent on its functionality (Vickers and Remaley 2014, Kontush 2014). HDL particles are heterogeneous in their composition and function, and in the circulation they transport numerous different cargos. These cargo-substances are biologically important for the functionality of HDL (Vickers and Remaley 2014). Steroid fatty acyl esters are endogenous cargos of HDL. Our finding that DHEA fatty acyl esters in HDL enhanced the vasodilatory capacity of HDL (I) suggests that these cargos may have relevance in the vascular effects of HDL. Hence, DHEA fatty acyl esters may improve antiatherogenic function of HDL. However, we used supraphysiological concentrations (10 μmol/l) of DHEA during the plasma incubation, and thus HDL presumably contained higher than normal DHEA fatty acyl ester concentrations. In addition to DHEA esters synthesized

52

during the incubation, isolated HDL particles contained an unknown amount of endogenous DHEA esters present in female HDL, as did also the native HDL. HDL enriched with estradiol fatty acyl esters also caused a dose- dependent relaxation, which was significantly stronger than the relaxation caused by native HDL at the HDL protein concentrations of 1, 10, and 40 μg/ml, but not with the largest dose 125 μg/ml (I). The relaxation response to estradiol ester-enriched HDL was not as strong as the response to DHEA ester-enriched HDL. However, DHEA becomes esterified and associated with lipoproteins at much higher rate than estradiol (Jones and James 1985, Helisten et al. 2001), and thus direct comparisons between the HDLs enriched with DHEA esters and estradiol esters are difficult to make. Previous work by Gong and co-workers (2003) suggests that HDL- associated estradiol may play a role in eNOS activation and vasodilation caused by HDL. They demonstrated that HDL from female but not from male mice caused vasodilation in isolated mouse arterial rings. HDL from premenopausal women and postmenopausal women receiving estrogen therapy induced eNOS activation in endothelial cells, whereas HDL from postmenopausal women without estrogen therapy or HDL from men did not stimulate eNOS. Furthermore, Gong and co-workers (2003) showed that endothelial cells could internalize HDL-associated estradiol. In the HDL fractions causing eNOS activation, physiological estradiol concentrations were measured. They suggested that estradiol associated with HDL could mediate eNOS activation caused by HDL. However, estradiol fatty acyl esters were not analyzed, and based on previous studies, estradiol is known to be associated with HDL in fatty acyl esterified form (Leszczynski and Schafer 1991, Höckerstedt et al. 2002, Vihma et al. 2003a). In contrast to the findings by Gong et al., Nofer and co-workers (2004) found no difference between vasodilatory responses to male and female HDL in mouse aortic rings. Our group has previously reported that increased serum estradiol fatty acyl ester concentrations during postmenopausal hormone therapy correlate positively with enhanced forearm blood flow responses in vivo (Vihma et al. 2003b). Our present finding of slightly enhanced vasodilation by estradiol fatty acyl ester-enriched HDL together with our previous findings in vivo (Vihma et al. 2003b) suggest that estradiol fatty acyl esters may also contribute to the vasodilatory function of HDL. Another cargo of HDL, namely sphingosine-1-phosphate (S1P), induces vasodilation, and the vasodilatory effect of HDL is proposed to be partly mediated by S1P (Nofer et al. 2004, Roviezzo et al. 2006, Egom et al. 2013). Hence, there are many cargos, including steroid fatty acyl esters and S1P, which may contribute to the positive effects of HDL on endothelial function independent of the plasma HDL cholesterol level. The protective effect of HDL against cardiovascular disease may depend on these and other cargos influencing the functionality of HDL.

53 Discussion

6.3.2 RECEPTORS INVOLVED IN DHEA FATTY ACYL ESTER- INDUCED VASODILATION HDL enriched with DHEA fatty acyl esters induced relaxation in isolated rat arterial rings via the SR-BI receptor pathway (II). Also native HDL caused vasodilation through SR-BI (II), consistent with previous studies. HDL binding to SR-BI is known to enhance eNOS activity and endothelium- dependent vasodilation (Yuhanna et al. 2001). In the endothelium, SR-BI and eNOS are colocalized in specific plasma membrane domains called caveolae (Uittenbogaard et al. 2000, Yuhanna et al. 2001). HDL activates, in SR-BI-mediated pathway, complex cellular signaling cascades leading to the phosphorylation of eNOS at serine-1177, which activates the enzyme (Mineo et al. 2003). Simultaneous cholesterol flux is required for the signal initiation by HDL–SR-BI interaction (Saddar et al. 2010). By inhibiting SR-BI- mediated lipid transport with a potent inhibitor, BLT-1, the relaxation response to DHEA ester-enriched HDL was dramatically attenuated (II). This led us to hypothesize that HDL-incorporated DHEA fatty acyl esters could be internalized by endothelial cells through SR-BI-mediated selective uptake and hydrolyzed to free DHEA, a potent vasodilator. In cultured human endothelial cells, we showed that HDL-associated DHEA fatty acyl esters were internalized partly via SR-BI, and after the internalization DHEA fatty acyl esters were hydrolyzed generating free DHEA (II). This process was, however, slow, proceeding during several hours, whereas relaxation of the arterial rings occurred in minutes. Therefore, it is unlikely that the liberated DHEA would be responsible for the rapid vasodilatory action of DHEA ester-enriched HDL. The vasodilatory effect of DHEA fatty acyl ester-enriched HDL was not mediated by androgen or estrogen receptors (II). This indicates that the relaxation by DHEA ester-enriched HDL was not facilitated by the conversion of DHEA to biologically active androgens or estrogens. This was not unexpected, as the relaxation response was very rapid, appearing immediately after the addition of DHEA ester-enriched HDL (I, II). Free, unesterified DHEA has been shown to rapidly activate eNOS in endothelial cells independently of androgen and estrogen receptors (Liu and Dillon 2002, Simoncini et al. 2003). It has been suggested that DHEA may have a specific plasma membrane receptor mediating eNOS activation in vascular endothelium (Liu and Dillon 2002, Liu and Dillon 2004). In ovariectomized rats with aortic banding-induced pressure overload, DHEA treatment enhanced aortic eNOS expression as well as phosphorylation of Akt and eNOS through sigma-1 receptor (Bhuiyan et al. 2011). In our study, however, the vasodilation was induced by DHEA fatty acyl ester-enriched HDL binding to SR-BI (II), and was probably not mediated through conversion of DHEA esters to free DHEA.

54

6.3.3 ROLE OF ENDOTHELIAL NITRIC OXIDE SYNTHASE IN DHEA FATTY ACYL ESTER-INDUCED VASODILATION HDL activates eNOS and nitric oxide production in cultured endothelial cells (Yuhanna et al. 2001, Li et al. 2002, Mineo et al. 2003, Nofer et al. 2004). Moreover, HDL causes rapid relaxation in isolated aortic rings of rats and mice, and pretreatment of the rings with eNOS antagonist L-NAME abolishes this effect (Yuhanna et al. 2001, Nofer et al. 2004). In our study, L-NAME markedly impaired the relaxation response to DHEA fatty acyl ester-enriched HDL, suggesting this response to be partly mediated by eNOS and nitric oxide (I). In addition, pretreatment of the arteries with L-NAME weakened the vasodilatory effect of native HDL, but the difference did not reach statistical significance (I). To further explore the role of eNOS in vasodilation caused by DHEA ester-enriched HDL, we continued our studies in human vascular endothelial cells. We demonstrated that DHEA fatty acyl ester-enriched HDL caused serine-1177 phosphorylation of eNOS (Paatela H et al. unpublished results). This effect was observed as early as in 5 min and reached the maximum at 10 min, when the amount of phosphorylated eNOS in the cells treated with DHEA ester-enriched HDL was nine times as high as in the cells treated with the buffer. Previous studies using endothelial cell cultures have shown that HDL activates eNOS through phosphorylation of serine-1177 residue (Mineo et al. 2003, Nofer et al. 2004, Kimura et al. 2010). HDL stimulates phosphorylation of Akt kinase, which in turn phosphorylates eNOS, leading to the activation of the enzyme and production of nitric oxide (Mineo et al. 2003, Nofer et al. 2004). Phosphorylation of eNOS at serine-1177 and parallel activation of MAP kinase pathway have been suggested to be required for the activation of eNOS by HDL (Mineo et al. 2003). Consistent with the previous studies, we also demonstrated a rapid serine-1177 phosphorylation of eNOS by native HDL (Paatela H et al. unpublished results). In addition, we showed that DHEA ester-enriched HDL caused eNOS phosphorylation, supporting the role of eNOS and nitric oxide in the vasodilatory effect of DHEA ester-enriched HDL. However, effects of DHEA ester-enriched HDL and native HDL on eNOS phosphorylation did not significantly differ, and thus the phosphorylation of eNOS in endothelial cells does not explain the difference in the arterial relaxation responses caused by these HDLs. The semiquantitative Western blot analysis of phosphorylated eNOS protein is probably not sensitive enough to detect small differences in the phosphorylation state of the enzyme. Furthermore, endothelial cell culture is not comparable to the intact arterial rings that contain also smooth muscle cells, which are eventually causing the vasodilation. There is evidence that HDL has vasodilatory activity not only through nitric oxide release, but also through production of prostacyclin (Norata et al. 2004, Zhang et al. 2012). In addition to the production of nitric oxide, its inactivation is an important factor influencing the bioavailability of vascular nitric oxide (Förstermann 2010). Furthermore, there are additional eNOS

55 Discussion phosphorylation sites, other than serine-1177, that affect eNOS activity (Fleming 2010). Increased production of prostacyclin, reduced inactivation of nitric oxide, or changes in the phosphorylation state of other residues of eNOS could be additional mechanisms for enhanced vasodilatory activity of DHEA ester-enriched HDL. These mechanisms, however, were not investigated in the present study.

6.3.4 ENDOTHELIAL UPTAKE AND METABOLISM OF HDL- ASSOCIATED DHEA FATTY ACYL ESTERS In Study II, we demonstrated that human umbilical vein endothelial cells internalized HDL-associated DHEA fatty acyl esters. This uptake was partly mediated by HDL receptor SR-BI function. Lipoprotein-associated DHEA fatty acyl esters are internalized and metabolized by different types of cultured cells. Human breast cancer cells have been shown to internalize LDL-associated DHEA fatty acyl esters via LDL receptor-mediated uptake and to further metabolize DHEA esters to DHEA and androstenediol (Roy and Bélanger 1993). In addition, human HeLa cells, derived from reproductive tract, are able to internalize LDL-associated DHEA fatty acyl esters through LDL receptor or LDL receptor-related receptor pathway, and after the internalization, DHEA esters are hydrolyzed and metabolized (Wang F et al. 2008). The present study was the first to demonstrate that human endothelial cells can internalize lipoprotein-associated DHEA fatty acyl esters. A significant proportion of DHEA circulates in fatty acyl esterified form incorporated in lipoprotein particles (Roy and Bélanger 1993), and thus lipoprotein-transported DHEA fatty acyl esters may be important precursors of active hormones in target tissues. Cellular uptake of HDL-associated DHEA by endothelial cells could potentially modulate vascular function. HDL receptor SR-BI appears to be active in the uptake of HDL-associated DHEA fatty acyl esters by endothelial cells, but other routes may participate, as well (II). Supporting the role of SR-BI in steroid fatty acyl ester uptake, this receptor has been shown to internalize HDL-associated estradiol fatty acyl esters in cultured hepatic cells (Badeau RM et al. 2007) and human macrophages (Badeau RM et al. 2009). Furthermore, SR-BI participates in the uptake of estradiol associated with HDL and LDL by osteoblastic cells (Brodeur et al. 2008). After the internalization of HDL-associated DHEA fatty acyl esters, endothelial cells were able to hydrolyze them. The cells further secreted liberated DHEA and unidentified polar metabolites of DHEA to the cell culture medium. This was, however, observed after several hours of incubation of the cells with DHEA fatty acyl ester-enriched HDL (II). Steroid fatty acyl esters are generally regarded as inactive hormone derivatives, which need to be hydrolyzed before binding to steroid receptors and exerting hormonal actions (Hochberg 1998). hydrolyzing steroid fatty acyl esters are not fully known. Studies in bovine placental and adipose tissue

56

have suggested that hormone-sensitive lipase may be the enzyme hydrolyzing estradiol fatty acyl esters (Lee et al. 1988). In human adipose tissue, hormone-sensitive lipase hydrolyzes DHEA and cholesteryl esters and is partly responsible for the hydrolysis of estradiol fatty acyl esters (Wang F et al. 2012). In human HeLa cells, lysosomal acid lipase participates in the hydrolysis of DHEA fatty acyl esters, but other unknown esterases are also involved (Wang F et al. 2008). Thus, different tissues may have distinct steroid fatty acyl ester hydrolyzing activities. Our study demonstrated that human endothelial cells were able to hydrolyze DHEA fatty acyl esters (II). We further showed that other metabolites of DHEA were secreted from the cells (II), suggesting existence of steroid-converting enzyme activity. DHEA, as a hormone precursor, can be converted to androgens and estrogens in peripheral target tissues (Labrie et al. 2005). Previous studies have shown that cultured breast cancer cells can convert LDL-associated DHEA esters to DHEA and androstenediol (Roy and Bélanger 1993), and HeLa cells can metabolize LDL-associated DHEA esters to DHEA, androstenedione, and 5α-androstanedione (Wang F et al. 2008). Thus, circulating DHEA fatty acyl esters associated with lipoproteins may serve as substrates for peripheral steroid hormone synthesis. By pretreating the endothelial cells with BLT-1, a potent inhibitor of SR-BI-mediated lipid transport, the secretion of free DHEA from the cells was markedly reduced (II). This suggests that SR-BI could mediate the flux of unesterified DHEA, like that of cholesterol (Mineo and Shaul 2012), from endothelial cells. Although cultured endothelial cells are not comparable to intact vascular endothelium, our data suggest that cellular hydrolysis of DHEA esters by endothelial cells is a relatively slow reaction, and thus it is unlikely that the liberated DHEA would be responsible for the rapid vasodilatory effect of DHEA ester-enriched HDL in the vascular wall. Another possibility is that DHEA fatty acyl esters incorporated in the HDL particle somehow modify the structure and function of HDL itself and thus enhance its vasodilatory capacity.

6.4 DHEA METABOLISM IN ADIPOSE TISSUE

After the cessation of ovarian estrogen production, all estrogens are synthesized in peripheral tissues from hormone precursors (Labrie 2015). In postmenopausal women, adipose tissue is an important source of estrogens. Our study demonstrated that steroid sulfatase activity, measured as the conversion of DHEAS to DHEA, in subcutaneous and visceral adipose tissue was higher in postmenopausal than in premenopausal women (III). This suggests that DHEAS, which circulates at much higher concentration than any other steroid hormone, could be more efficiently utilized by postmenopausal compared to premenopausal adipose tissue. Thus, the

57 Discussion hydrolysis of DHEAS in adipose tissue may play a more important role in steroid biosynthesis after menopause. Adipose tissue expresses all the enzymes necessary for the formation of active androgens and estrogens from prohormones DHEA and DHEAS (Tchernof et al. 2015, Li et al. 2015). We investigated gene expression of some key enzymes related to the formation of estradiol. The mRNA expression levels of aromatase (CYP19A1 gene) and 17βHSD type 12 were higher in postmenopausal than in premenopausal women in subcutaneous adipose tissue. In visceral fat, the mRNA expression of hormone-sensitive lipase (LIPE gene) was higher in postmenopausal than in premenopausal women. Aromatase is responsible for the conversion of androstenedione to estrone and testosterone to estradiol (Forney et al. 1981). Type 12 17βHSD is possibly the most important enzyme of the 17βHSD family in converting estrone to biologically active estradiol in adipose tissue (Bellemare et al. 2009). Hormone-sensitive lipase participates in the hydrolysis of DHEA and estradiol fatty acyl esters (Wang F et al. 2012). As all these three enzymes drive towards the formation of biologically active estradiol, our results are in line with the possibility that production of estrogens in adipose tissue could be enhanced after menopause. Steroid sulfatase activity was higher in visceral than in subcutaneous adipose tissue, analyzed in premenopausal women or in all women. There are no previous studies comparing sulfatase activity between different adipose tissue depots. In fact, steroid sulfatase activity has been previously reported only in human subcutaneous adipose tissue (Dalla Valle et al. 2006). Accumulation of visceral adipose tissue is related to the development of metabolic syndrome, type 2 diabetes, and cardiometabolic and cardiovascular complications (Fox et al. 2007, Smith et al. 2012). Sex hormones are important regulators of regional fat accumulation (Tchernof and Després 2013). Men and women display different body fat distribution patterns, and after menopause, visceral adipose tissue increases (Genazzani and Gambacciani 2006, Lovejoy et al. 2008, Tchernof and Després 2013). Our finding of different DHEA sulfatase activity in subcutaneous and visceral adipose tissue depots suggests that body fat distribution may have relevance in modulating hormone metabolism, or conversely, local hormone metabolism may influence body fat distribution. Consistent with previous studies, subcutaneous adipose tissue showed a higher mRNA expression of steroid sulfatase, aromatase and hormone- sensitive lipase compared to visceral adipose tissue. Previous studies in adipose tissue obtained from women and obese men have shown that STS gene is more expressed in subcutaneous than in visceral fat (Blouin et al. 2009a, Wang F et al. 2013). Also aromatase mRNA expression is reported to be higher in subcutaneous adipose tissue (Wang F et al. 2013), although other studies have found only slightly higher expression in subcutaneous compared to visceral fat (Blouin et al. 2006, Blouin et al. 2009a). In addition, previous studies show that hormone-sensitive lipase displays higher mRNA

58

expression in subcutaneous than in visceral adipose tissue (Ray et al. 2009, Wang F et al. 2013) and adipocytes (Reynisdottir et al. 1997). Other studies, however, have found no difference in the mRNA expression of hormone- sensitive lipase in adipose tissue (Lefebvre et al. 1998) or adipocytes (Montague et al. 1998) derived from different fat depots. In contrast to previous studies (Bellemare et al. 2009, Blouin et al. 2009b, Wang F et al. 2013), we detected a higher mRNA expression of 17βHSD type 7 in visceral than in subcutaneous adipose tissue. Depot-differences in the steroid sulfatase activity as well as in the gene expression of steroid-converting enzymes found in our study and previous studies suggest that steroid hormone synthesis and metabolism may differ between adipose tissue depots. Steroid sulfatase activity was higher in visceral adipose tissue, whereas mRNA expression of STS was higher in subcutaneous fat. Furthermore, DHEAS was more efficiently hydrolyzed in postmenopausal than in premenopausal adipose tissue, but STS mRNA expression did not differ between the groups. These differences may be explained by the possibility that steroid sulfatase is regulated by posttranscriptional and posttranslational modifications (Mueller et al. 2015). The regulation of steroid sulfatase activity has not been studied in adipose tissue. In breast cancer cells, inflammatory cytokines have been shown to enhance steroid sulfatase activity (Purohit et al. 1996, Newman et al. 2000, Honma et al. 2002), and this activation occurs without increase in STS mRNA expression (Newman et al. 2000). Postmenopausal women have an increased inflammatory response in abdominal (Alvehus et al. 2012) and breast (Iyengar et al. 2015) subcutaneous adipose tissue. Furthermore, adipose tissue distribution influences inflammatory cytokine secretion (Tchernof and Després 2013). Our findings of higher sulfatase activity but similar STS mRNA expression after menopause could be explained by increased adipose tissue inflammation in postmenopause. Inflammatory factors might also explain the different profiles of steroid sulfatase activity and mRNA expression between adipose tissue depots. The mRNA expression levels of the genes studied did not correlate with serum concentrations of DHEAS or DHEA. Serum estrone and estradiol, however, correlated positively with the adipose tissue STS mRNA expression in postmenopausal women. No such correlation was found in premenopausal women, as expected due to the fluctuation in serum estrogen levels during the menstrual cycle. Our finding suggests that steroid sulfatase in adipose tissue may contribute to serum estrogen levels after menopause, when all estrogens derive from peripheral tissues. Steroid sulfatase activity did not correlate with serum estrogen concentrations. However, we measured steroid sulfatase activity by the conversion of DHEAS to DHEA and not by the conversion of estrone sulfate to estrone, which might have yielded a different result.

59 Discussion

In all women analyzed together, steroid sulfatase activity correlated positively with the mRNA expression levels of steroid sulfatase, aromatase, and 17βHSD type 12 in subcutaneous adipose tissue. This raises the possibility that enhanced hydrolysis of DHEAS could be related to the increased expression of these genes, further directing this pathway towards the formation of biologically active estradiol. Sulfation and desulfation of steroid hormones are important regulatory mechanisms in steroidogenesis and hormone actions in peripheral tissues (Mueller et al. 2015). Sulfated steroids, such as DHEAS and estrone sulfate, are suggested to act as reservoirs of substrates for the peripheral synthesis of biologically active steroid hormones (Mueller et al. 2015). DHEAS concentrations in the circulation are reported to be much higher than those in adipose tissue (Deslypere et al. 1985), whereas other sex steroids, including DHEA, estradiol, and estrone, are more abundant in adipose tissue than in serum (Deslypere et al. 1985, Badeau M et al. 2007, Wang F et al. 2011, Kinoshita et al. 2014, Vihma et al. 2016). Low DHEAS concentrations in adipose tissue may be due to active and rapid hydrolysis of this hydrophilic derivative in the tissue. Circulating DHEAS can be internalized by adipocytes through an active transport, presumably facilitated by OATP (Dalla Valle et al. 2006). Our study suggests that adipose tissue could utilize DHEAS, derived from the circulation, more efficiently after menopause. Thus, more DHEA could be liberated for further conversion to biologically active androgens and estrogens.

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7 SUMMARY, CONCLUSIONS, AND FUTURE PERSPECTIVE

The present thesis aimed at elucidating the role of DHEA in lipophilic environment – circulating lipoproteins and adipose tissue. The results can be summarized as follows:

1) DHEA was esterified to its fatty acyl ester derivatives, when incubated with human plasma, and these lipophilic derivatives were incorporated in plasma lipoproteins.

2) DHEA fatty acyl ester-enriched HDL showed a stronger vasodilatory effect compared to native HDL in isolated rat arterial rings ex vivo. The vasodilatory effect of DHEA ester-enriched HDL was mediated by HDL receptor SR-BI, but not through androgen or estrogen receptors. The arterial relaxation was in part mediated by eNOS. DHEA ester-enriched HDL and native HDL induced rapid eNOS phosphorylation in cultured endothelial cells. These results suggest that DHEA fatty acyl esters, associated with HDL as cargos, enhance the vasodilatory capacity of HDL and thus may improve the antiatherogenic function of HDL. This is a novel biological function for lipoprotein-associated steroid fatty acyl esters.

3) Human vascular endothelial cells were able to internalize HDL-associated DHEA fatty acyl esters. This uptake was partly mediated by SR-BI. After the cellular internalization, DHEA esters were metabolized to DHEA and unknown polar metabolites, which were further secreted from the cells. These results suggest a role for lipoprotein-associated DHEA fatty acyl esters as substrates in peripheral steroid hormone synthesis. Cellular uptake and hydrolysis were slow, and thus it is unlikely that the liberated DHEA would be responsible for the rapid vasodilatory effect of DHEA ester-enriched HDL.

4) Steroid sulfatase activity, the conversion of DHEAS to DHEA, in abdominal subcutaneous and visceral adipose tissue was higher in postmenopausal than in premenopausal women. Visceral adipose tissue showed a higher sulfatase activity compared to subcutaneous adipose tissue. Three genes in the estradiol-producing pathway, aromatase, 17βHSD type 12, and hormone-sensitive lipase, were more expressed in postmenopausal compared to premenopausal adipose tissue. These results suggest that circulating DHEAS could be more efficiently utilized in postmenopausal adipose tissue for the formation of biologically active androgens and estrogens. Depot-differences in the sulfatase activity and in the gene expression of steroid-converting enzymes suggest that steroid hormone metabolism may differ between adipose tissue depots.

61 Summary, conclusions, and future perspective

There are certain aspects to be considered in future research. The HDL- mediated cardioprotection is thought to be dependent on the functionality of HDL and not on the HDL cholesterol level. Hence, the HDL cholesterol hypothesis has recently been replaced with the HDL function hypothesis. HDL transports numerous biologically active substances, which are important for the functionality of HDL. Our study demonstrates that steroid hormones, also cargos of HDL, may have relevance in the vasodilatory effect of HDL. Thus, they may act as endothelotherapeutic agents enhancing the positive effects of HDL. Future studies are crucial to elucidate whether HDL- associated steroid hormones have influence on other protective effects of HDL, including anti-inflammatory, antioxidative, and antithrombotic activities. In addition, the mechanisms by which steroid hormones modulate the function of HDL need to be further clarified. Regarding the role of adipose tissue in the steroid biosynthesis, future studies are necessary to clarify to what extent steroid sulfatase hydrolyzes estrone sulfate and whether other steroid-converting enzymes in the metabolic pathway involved in the formation of androgens and estrogens are active in human subcutaneous and visceral adipose tissue. Moreover, of special interest would be to elucidate whether biologically active androgens and estrogens synthesized in adipose tissue only act locally in the same tissue or are transferred to the circulation.

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8 ACKNOWLEDGEMENTS

This thesis project was carried out in the Preventive Medicine Research Program of the Folkhälsan Research Center and in the Heart and Lung Center, University of Helsinki and Helsinki University Hospital. This work was financially supported by the Folkhälsan Research Center, the Sigrid Jusélius Foundation, the Päivikki and Sakari Sohlberg Foundation, the Finnish Foundation for Cardiovascular Research, Finska Läkaresällskapet, and the State funding for university-level health research. I express my deepest gratitude to my supervisors, Professor Matti J. Tikkanen and Adjunct Professor Matti Jauhiainen. Professor Tikkanen has provided excellent working facilities and introduced me to the field of research. He has always found time to discuss any possible problems in my work and given encouraging advice and ideas. Adjunct Professor Jauhiainen has been extremely optimistic and has never run out of ideas during these years. I admire his technical expertise, energetic approach, and endless enthusiasm towards science. I am deeply grateful to my supervisors for their guidance and encouragement, and for understanding my life priorities. I acknowledge the Director of the Folkhälsan Research Center, Professor Anna-Elina Lehesjoki, for providing excellent research facilities and working environment in Biomedicum Helsinki. I also want to express my gratitude to Professor Herman Adlercreutz. He was an exceptional researcher, dedicated to science, and a role model for young researchers. The official reviewers, Professors Matti Poutanen and Raimo Voutilainen, are acknowledged for their careful evaluation of this thesis and their constructive comments and advice. I want to thank the members of my thesis committee, Professor Vesa Olkkonen and Adjunct Professor Katariina Öörni for showing interest in and support for this thesis project. I am grateful to Professor Eero Mervaala for important collaboration. I appreciate his vast expertise, innovative ideas, and encouraging attitude. He provided me an opportunity to work with the excellent technicians of his laboratory, Anneli von Behr and Nada Bechara-Hirvonen. I owe my deepest gratitude to Anneli and Nada for their great effort in the relaxation studies and the cell culture work, and also for their kindness and support. I acknowledge Adjunct Professor Tomi Mikkola for collaboration in the adipose tissue project, Adjunct Professors Esa Hämäläinen and Ursula Turpeinen for their expertise in the steroid hormone analyses, and Professor Kristiina Wähälä and her team including Somdatta Deb for synthesizing steroid standards that were essential to the work. I am deeply grateful to Veera Vihma for the collaboration and especially for her important help in the preparation of the second manuscript during my maternity leave. Veera, thank you for your friendship, your support and encouragement, your fantastic company during congress trips, and for

63 Acknowledgements sharing with me both the joyful and depressing moments during this thesis project. I am truly grateful to Feng Wang for her expertise and effort in analyzing the gene expressions in the adipose tissue project, and even more grateful for her friendship. Feng, my dear friend with a big heart, thank you for always being there for me. I want to thank Hanna Savolainen-Peltonen for fruitful collaboration, for numerous problem-solving sessions during our “Raiders of the Lost Nitric Oxide”-project, and for her support and friendship. I have been privileged to work with experienced laboratory technicians. I thank Kirsti Räsänen for extremely important help during these years. Kirsti, working with you has been a delight. Thank you for your expertise, support, and friendship. I also want to thank Päivi Ihamuotila for excellent technical assistance, interesting conversations, friendship, and for creating a positive and energetic atmosphere in our laboratory. My warmest appreciation goes to Terhi Hakala for teaching me all the basics in the laboratory work during the early phases of this work, to Päivi Ruha for the first-class technical assistance, and to Anne Ahmanheimo, Adile Samaletdin, and Anja Koskela for their support and encouragement. I warmly thank the present and former members of our lab, Natalia Bogdan, Päivi Söderholm, Maija Badeau, Anna Höckerstedt, Robert Badeau, and others, for making the work enjoyable. Of the colleagues in the neighboring groups, I want to thank Sanni Söderlund and Anna Ketomäki for their support and friendship. All the people at the Folkhälsan Research Center and in the cardiovascular research groups are warmly thanked for creating a pleasant working atmosphere. I thank nurses and doctors at the Women’s Hospital and the Kätilöopisto Maternity Hospital for their invaluable help in collecting samples. I express my gratitude to all the study subjects who volunteered for this study. I am grateful to all my wonderful friends – girls from the Sibelius high school, ladies from the medical school (the book club), friends of our family – for support, joyful moments, laughter, and even occasional tears. I owe my dearest thanks to my parents Stina and Pekka as well as my brothers Jarkko and Lauri, my sister Milka, and their families for their love and support. I thank my parents-in-law Marjatta and Risto for helping and caring, for always being there for us. Finally, I thank my beloved husband Teemu and our daughters Ellen, Linnea, and Sofia for their unconditional love, for encouragement and support, for our happy life, and for keeping my priorities straight. You are my everything.

Helsinki, April 2016

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