THYROID HORMONE DEIODINATION PROEFSCHRIFT ter verkrijging van de graad van doctor in de Geneeskunde aan de Erasmus Universiteit Rotterdam op gezag van Rector Magnificus Prof.Dr. J. Sperna Weiland en volgens besluit van het College van Dekanen. De openbare verdediging zal plaatsvinden op woensdag 22 oktober 1980 des namiddags te 2.00 uur door THEOFILUS JOHANNES VISSER geboren te Rotterdam Promotor: Prof.Dr. G. Hennemann Coreferenten: Prof.Dr. W.C. HU:lsmann Prof.Dr. H.J. van der Molen Aan Rian, Ellen en Renske. CONTENTS page VOORWOORD 9 LIST OF ABBREVIATIONS 10 SCOPE OF THE THESIS 11 Chapter L GENERAL INTRODUCTION 13 1. 1. Thyroid hormone biosynthesis 13 1.2. Thyroid hormone action; structure-activity relationship 17 1. 3. Pathways of thyroid hormone metabolism 18 1.4. Conclusions 21 Chapter 2. THYROID HORMONE DEIODINATION - IN VIVO OBSERVATIONS 23 2. L Introduction 23 2.2. Deiodination under physiological conditons 24 2.3. Factors affecting deiodination 28 2.3.1. Inhibitors 28 2.3.1.1. Thiouracil derivatives 28 2. 3.1. 2. s-Adrenergi c antagonists 29 2.3.1.3. Glucocorticosteroids 30 2.3.1.4. Miscellaneous 31 2.3.2. Diet 34 2.3.3. Pathological conditons 38 2.3.3.1. Stress 38 2.3.3.2. Illness 39 2.4. Conclusions 40 Chapter 3. THYROID HORI~ONE OEIODINATION - IN VITRO OBSERVATIONS 42 3.1. Introduction 42 3.2. Degradation of iodothyronines with release of iodide 42 3.3. Production of specific metabolites 46 3.4. localization of deiodinase activity 48 3.5. Characteristic features of enzymatic deiodination 49 3.6. Cofactors 54 3.7. Inhibitors 55 3.8. Possible mechanisms of deiodination 58 3.9. Conclusions 63 Chapter 4. REGULATION OF THYROID HORMONE DEIODINATION 66 4.1. Introduction 66 4.2. Effects of nutritional and thyroid status 66 4.3. General conclusions 70 REFERENCES 73 SUMI1ARY 91 SAMENVATTING 93 CURRICULUM VITAE 96 APPENDIX PAPERS 97 I. T.J. Visser, I. van der Does-Tobe, R. Docter and G. Hennemann: Conversion of thyroxine into triiodothyronine by rat liver homogenate. Biochem. J. l§Q, 489-493 (1975). II. T.J. Visser, I. van der Does-Tab&, R. Docter and G. Hennemann: Subcellular localization of a rat liver enzyme converting thyroxine into triiodothyronine and possible involvement of essential thiol groups. Bi ochem. J. ~- 479-482 ( 1976). III. T.J. Visser, D. Fekkes, R. Docter and G. Hennemann: Kinetics of enzymic reductive deiodination of iodothyronines; effect of pH. Biochem. J. ~- 489-495 (1979). IV. T.J. Visser: Mechanism of action of iodothyronine 5'-deiodinase. Biochim. Biophys. Acta 569, 302-308 (1979). V. T.J. Visser: r~echanism of inhibition of iodothyronine 5'-deiodinase by thioureylenes and sulfite. Biochim. Biophys. Acta~- 371-378 (1980). VI. T.J. Visser and E. van Overmeeren: Substrate requirement for inactivation of iodothyronine 5'-deiodinase activity by thiouracil. (submitted for publication). 9 VOORWOORD Het onderzoek dat resulteerde in de bewerking van dit proefschrift werd verricht op de afdeling Inwendige Geneeskunde III, tevens afdeling voor Klinische Endocrinologie en Stofwisselingsziekten (hoofd: Prof.Dr. J.C. Birken­ hager), van het Academisch Ziekenhuis Dijkzigt te Rotterdam. Velen hebben mij gesteund bij de uitvoering van de experimenten en het schrijven van dit proef­ schrift. Met name wil ik danken: Mijn promotor, Jorg Hennemann, voor de stimulerende discussies, ZlJO belang­ stell ing voor de voortgang van mijn onderzoek en de vrijhei d die hij mij 1 iet bij het bepalen van de onderzoekslijn; - De co-referenten, Wim HUlsmann en Henk van der ~1olen, voor hun uiterst waarde­ volle kommentaren op mijn manuscript; Reel Docter, voor zijn adviezen en zorg voor de technische aspekten van mijn werk, met name de ontwikkeling en uitvoering van de radioimmunoassays; - Durk Fekkes, Eric Krenning, Steven Lamberts, Marten Otten en Paul Wilson, voor het genoegen met hen te discussieren over en samen te werken aan het hier beschreven onderzoek; - Jasper Scholte, voor zijn adviezen betreffende de subcellulaire fraktionering; - De medewerkers van het laboratorium, voor de goede sfeer en de plezierige samenwerking; In het bijzonder Ineke van der Does en Ellen van Overmeeren voor de vaardige hulp bij de uitvoering van de experimenten met betrekking tot dit proefschri ft; - Corry Boot, voor het accuraat en snel uittypen van de manuscripten en de on­ misbare assistentie bij de bewerking van dit proefschrift; - De medewerkers van de grafische afdeling van de Audiovisuele Dienst voor de zorgvuldige uitvoering van de figuren. 10 LIST OF ABBREVIATIONS DIT 3,5-diiodotyrosine DTE dithioerythritol OTT dithiothreitol E- I enzyme-iodine complex with undefined oxidation state E-SH enzyme with free sulfhydryl group E-SI enzyme-sulfenyl iodide complex GSH reduced glutathione GSSG oxidized glutathione MCR metabolic clearance rate MIT 3-(mono)iodotyrosine M-SH methimazole; 2-mercapto-1-methylimidazole NEM N-ethylmaleimide PR production rate PTU 5- or 6-propyl-2-thiouracil R-SH reduced cofactor R-S-S-R oxidized cofactor rT reverse T ; 3,3' ,5'-triiodothyronine 3 3 To thyronine 3- or 3'-T1 3- or 3'-iodothyronine 3,5-, 3,3'- or 3' 3,5-, 3,3'- or 3' ,5'-diiodothyronine ,s·-r2 T3 3,3' ,5-triiodothyronine T4 thyroxine; 3,3' ,5,5'-tetraiodothyronine Tetrac 3,3',5,5'-tetraiodothyroacetic acid Tin i odothyroni ne T -binding globulin TBG 4 TBPA T4-binding prealbumin TRH TSH-releasing hormone Triac 3,3' ,5-triiodothyroacetic acid TSH thyroid-stimulating hormone; thyrotropin TU 2-thiouracil (also: U-SH) U-SH 2-thiouracil (also: TU) 11 SCOPE OF THE THESIS The enzymatic deiodination of thyroid hormone is an important process since it concerns- among other things- the regulation of thyromimetic act­ ivity at the site of the target organ. To understand the mechanism of this regulation it is necessary to have a detailed knowledge of the mode of action of the enzyme(s) involved in the metabolism of thyroid hormone. My investigat­ ions of the deiodination of iodothyronines at the subcellular level, forming the basis of this thesis, are described in the appendix papers. It is not intended to deal in extenso with the technical aspects of my studies in the preceeding chapters. Rather it will be attempted to give a general review of the literature including- with some emphasis -my own work. Though not directly related to the subject of this thesis, the bio­ synthesis of thyroid hormone in the thyroid gland is treated in the first chapter. This is done because of possible similarities between thyroid hormone iodination and deiodination pathways, which are suggested by the finding that some drugs inhibit both processes. In the same chapter the relationship between iodothyronine structure and biological potency is described to illu­ strate that indeed deiodination has a dramatic effect on the activity of thyroid hormone. Besides deiodination, other pathways of metabolism are also considered. The second chapter concerns the in vivo investigation of thyroid hormone deiodination under physiological and pathological conditions. This includes the effects of internal and external factors which affect deiodination, such as dietary intake, drugs, stress and illness. Since much work has been done to find an explanation for the effect of calorie restriction on deiodination at the molecular level, the role of the diet is emphasized. This appears particularly important since nutritional status must be considered to contrib­ ute to the change in thyroid hormone metabolism found in other situations, for example in systemic illness. The in vitro observations of the enzymatic deiodination of thyroid hormone are described in chapter 3. A distinction has been made between (early) reports on the analysis of iodide production using chromatography, and (more recent) studies dealing with the detection of specific metabolites, often by means of radioimmunoassay. My investigations which belong to the latter category are presented in the appendix papers. In the last chapter an attempt is made to relate the in vivo findings (described in chapter 2) to the characteristic features of the enzymatic 12 reactions as revealed by the in vitro experiments (described in chapter 3). Especially the role of glutathione is emphasized with respect to changes in thyroid hormone metabolism during fasting. 13 1. GENERAL INTRODUCTION 1.1. Thyroid hormone biosynthesis The principal secretory products of the thyroid gland are thyroxine (3,3' ,5,5'-tetraiodothyronine, T ) and 3,3' ,5-triiodothyronine (T ). Under 4 3 physiological conditions and sufficient iodine intake they are synthesized in the human gland in a molar ratio of about 20:1. This is much higher than the ratio of their blood production rates (approximately 3:1), indicating an additional source for circulating r 3. It is now well established that the extra-thyroidal monodeiodination of r4 at the 5'-position (5'-deiodination) is a major route of T production. For the isomer, 3,3' ,5'-triiodothyronine 3 (reverse T , rT ), peripheral production by monodeiodination of T at the 3 3 4 5-position (5-deiodination) is even more important. Thyroidal secretion of rT3 and probably also of the lower substituted iodothyronines is almost negligible. Thus, in normal persons, total production rates for T4, T3 and rT amount to about 115, 45 and 30 nmol/day, where thyroidal secretion 3 accounts for 100, 20 and 6%, respectively {Chopra et al, 1978a; see Table 1.1.). Table 1.1. CONTRIBUTIONS OF THYROIDAL SECRETION AND PERIPHERAL PRODUCTION TO TOTAL PRODUCTION RATES OF T , T AND rT a 4 3 3 Iodothyronine Total production Thyroidal secretion Peripheral (nmol/day per 70 kg production body weight (%)) T4 115 115 (100) T3 45 9 ( 20) 36 (80) rT 3 30 2 ( 6) 28 (94) aData from Chopra, 1976; Chopra et al, l978a (see also section 2.2.). Thyroxine is produced by coupling of two diiodotyrosine residues within the thyroglobulin molecule. Thyroglobulin is a high molecular weight (~660 000 dalton) glycoprotein.