! " # $% &'((% ()*+,- .-. (,/'*,-.-, 0.1,'(, 00.1. 2332 Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in Medical Pharmacology presented at Uppsala University in 2002 ABSTRACT Nordquist, J. 2002. Semicarbazide-sensitive amine oxidase and vascular complications in diabetes mellitus. Biochemical and molecular aspects. Acta Universitatis Upsaliensis. Comprehensive summaries of Uppsala Dissertations from the Faculty of Medicine 1174. 51 pp. Uppsala ISBN 91-554-5375-9. Plasma activity of the enzyme semicarbazide-sensitive amine oxidase (SSAO; EC.1.4.3.6) has been reported to be high in disorders such as diabetes mellitus, chronic congestive heart failure and liver cirrhosis. Little is known of how the activity is regulated and, consequently, the cause for these findings is not well understood. Due to the early occurrence of increased enzyme activity in diabetes, in conjunction with the production of highly cytotoxic substances in SSAO-catalysed reactions, it has been speculated that there could be a causal relationship between high SSAO activity and vascular damage. Aminoacetone and methylamine are the best currently known endogenous substrates for human SSAO and the resulting aldehyde- products are methylglyoxal and formaldehyde, respectively. Both of these aldehydes have been shown to be implicated in the formation of advanced glycation end products (AGEs). This thesis is based on studies exploring the regulation of SSAO activity and its possible involvement in the development of vascular damage. The results further strengthen the connection between high SSAO activity and the occurrence of vascular damage, since type 2 diabetic patients with retinopathy were found to have higher plasma activities of SSAO and lower urinary concentrations of methylamine than patients with uncomplicated diabetes. From studies on mice, it was also found that an SSAO inhibitor potently reduces the incorporation of methylamine metabolites in the tissues. By quantifying SSAO-gene expression in alloxan-induced diabetes, increased transcription could be ruled out as a cause for the increased enzyme activity, thereby opening up for the possibility that the activity is regulated post-translationally. In fact, increased enzyme activity in adipose tissue was accompanied by decreased mRNA-levels, suggesting that the gene expression could be negatively controlled by the enzyme activity. Key words: SSAO, VAP-1, diabetes, retinopathy, methylamine, aminoacetone, hydralazine Jenny Nordquist, Department of Neuroscience, Unit of Pharmacology, Biomedical Center, Box 593, SE-751 24 Uppsala, Sweden © Jenny L.E. Nordquist 2002 ISSN 0282-7476 ISBN 91-554-5375-9 Printed in Sweden by Eklundshofs Grafiska, Uppsala 2002 2 A story of an enzyme Once upon a time, in the mid 20th century, researchers all over the world started to unravel the secrets of amine oxidising enzymes. One family of such enzymes was the semicarbazide-sensitive amine oxidases (SSAOs). The researchers found out a lot of things about SSAOs: There were enzymes belonging to this family in practically all living organisms, they characterised them regarding substrate-specificity and inhibitor-sensitivity, and they found that in humans, the activity was increased in pathological conditions such as diabetes mellitus, liver cirrhosis and chronic congestive heart failure. And yet, the researchers were troubled… They could not find that it had any conceivable physiological importance! In humans, they could not even find good endogenous substrates for the enzyme. Despite large efforts, they did not get any closer to solving the mystery, and eventually, the enzyme was almost forgotten, a small burning flame of interest kept alive by but a few… Until… one day in the 1990´s, a gene coding for SSAO was cloned. Not long after that, another group of researchers in another part of the world, cloned another gene – or so they thought. How astonished they were when they found that their vascular adhesion protein 1 (VAP-1) was actually an amine oxidase, namely SSAO. Now this caught a lot of attention! SSAO was important in guiding leukocytes to sites of inflammation! Meanwhile, it was also discovered that the enzyme could affect the uptake of glucose into the cells by affecting the glucose transporters GLUT1 and GLUT4. Finally, some plausible physiological functions for SSAO! Could there be others yet to be discovered? Could it, for example, be that the increased enzyme activity in diabetes was a defence-mechanism? Or could this be an explanation for the vascular damage associated with diabetes? New questions followed and the story still goes on… The Beginning! 3 MAIN REFERENCES This thesis is based upon the following papers, which will be referred to in the text by their roman numerals: I. Jenny L.E. Grönvall, Håkan Garpenstrand, Lars Oreland and Jonas Ekblom. Autoradiographic imaging of formaldehyde adducts in mice: possible relevance for vascular damage in diabetes. (1998) Life Sciences, vol 63, no 9, pp.759-768 II. Jenny L.E. Nordquist, Cecilia Berggård, Håkan Garpenstrand, Jonas Ekblom and Lars Oreland. Autoradiographic study on aminoacetone-metabolism by semicarbazide-sensitive amine oxidase in mice. Manuscript III. Jenny L.E. Grönvall-Nordquist, Lars B. Bäcklund, Håkan Garpenstrand, Jonas Ekblom, Britta Landin, Peter H. Yu, Lars Oreland and Urban Rosenqvist. Follow-up of plasma semicarbazide- sensitive amine oxidase activity and retinopathy in Type 2 diabetes mellitus. (2001) Journal of diabetes and its complications 15, pp. 250-256. IV. Jenny L.E. Nordquist, Camilla Göktürk and Lars Oreland. Semicarbazide-Sensitive Amine Oxidase (SSAO) Gene Expression in Alloxan-induced Diabetes in Mice. Submitted Reprints were made with permission from the publisher, Elsevier Science. 4 TABLE OF CONTENTS Page ABBREVIATIONS………………………………………………………… 6 INTRODUCTION…………………………………………………………. 7 Clinical findings……………………………………………………… 7 Enzyme classification………………………………………………… 8 Genetics……………………………………………………………….10 Molecular structure and distribution………………………………… 13 Enzyme reaction, substrates and products……………………………16 Physiological role……………………………………………………. 19 Toxicity…….………………………………………………………….22 Diabetes mellitus…………………………………………………….. 24 SSAO in diabetes……………………………………………………... 26 PRESENT INVESTIGATION……………………………………………... 27 Aims of the thesis…………………………………………………….. 27 Article I………………………………………………………………. 27 Article II……………………………………………………………… 31 Article III…………………………………………………………….. 33 Article IV…………………………………………………………….. 35 CONCLUSIONS…………………………………………………………… 39 GENERAL DISCUSSION AND FUTURE PERSPECTIVES……………..40 Possible benefits of SSAO……………………………………………. 40 Possible regulatory mechanisms……………………………………...41 Investigations ahead……………………………………………….… 42 ACKNOWLEDGEMENTS…………………………………………………43 REFERENCES…………………………………………………………….. 45 5 ABBREVIATIONS cDNA complementary DNA CMC carboxymethyl cellulose DAO diamine oxidase DNA deoxyribonucleic acid EDTA ethylenediaminetetraacetic acid Glut glucose transporter HPAO human placental amine oxidase 5-HT 5-hydroxytryptamine (serotonin) i.p. intraperitoneal i.v. intravenous Km Michaelis-Mentens constant MAO monoamine oxidase mRNA messenger RNA OD optical density PCR polymerase chain reaction RAO retinal amine oxidase RNA ribonucleic acid SSAO semicarbazide-sensitive amine oxidase VAP-1 vascular adhesion protein 1 Vmax maximum velocity 6 INTRODUCTION The focus of this thesis is on a group of enzymes, known as semicarbazide- sensitive amine oxidases (SSAO; EC.1.4.3.6). This is a family of enzymes found in a large variety of species, from prokaryotic bacteria to higher eukaryotes, including humans (for a review, see Blaschko, 1974). However, to cover all the aspects of all these members of the SSAO-family would be far beyond the scope of this thesis, and therefore the discussion will be restricted mainly to the mammalian enzymes, what they are thought to look like, what is known about their function, why they could be beneficial and why they could also be detrimental. Clinical findings The first enzymes belonging to the SSAO family were characterised already in the 1950´s (Bergeret et al., 1957; Hirsch, 1953) and many early investigations were focused on quantifying the activity in different physiological and pathological conditions. These early, and also more recent clinical studies have provided the basis for many of the investigations that are being conducted today, and therefore, I will begin with a brief summary of such findings. First of all, the plasma activity of SSAO is rather stable in healthy individuals, although children have a higher activity than adults (Tryding et al., 1969; Murphy et al., 1977; Boomsma et al., 1999). However, the activity is altered in some conditions. For example, the plasma SSAO activity is decreased in the first trimester of pregnancy (Lewinsohn and Sandler, 1982) as well as in patients with severe burns (Lewinsohn, 1977). In contrast, it is increased in the third trimester of pregnancy (Lewinsohn and Sandler, 1982), in adolesence (Murphy et al., 1977) and in cancer with metastases (Ekblom et al., 1999). In addition to this, the activity is high in some conditions frequently associated with some form of vascular complications, e.g. diabetes mellitus (Tryding et al., 1969; Garpenstrand et al., 1999), liver cirrhosis (McEwen and 7 Castell, 1967), and congestive heart failure (McEwen and Harrison, 1965; Boomsma et al., 1997; Boomsma et al., 2000). In fact, the plasma SSAO activity in diabetes complicated by retinopathy