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.-. (,/'*,-.-, 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 and vascular complications in 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 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 and , 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- 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 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 . 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 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 ! Meanwhile, it was also discovered that the enzyme could affect the uptake of 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 HPAO human placental amine oxidase 5-HT 5-hydroxytryptamine (serotonin) i.p. intraperitoneal i.v. intravenous

Km Michaelis-Mentens constant MAO 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 is higher than in diabetes without this complication (Garpenstrand et al., 1999; Article III). Very little is known of how the SSAO activity is regulated, and therefore we do not know why the plasma activity is sometimes altered, as described above. Since there are indications suggesting that high SSAO activity may have injurious effects on blood vessels, this has become one of the key issues in the field of SSAO, and one of the questions explored in this thesis.

Enzyme classification

Before discussing SSAO any further, the term needs a definition. What make SSAOs distinct from other amine oxidases? In fact, this is not an easy question to answer. In the literature, SSAO is sometimes referred to as an enzyme found for example in human plasma, and sometimes the term is used to define a group of enzymes. In addition to this, many different names have been used to describe SSAOs, eg. oxidase, plasma amine oxidase, and benzylamine oxidase. The reason for the somewhat chaotic classification of SSAOs is, first of all, that it is a very diverse group of enzymes found in many species, and most likely with very diverse functions. For example, SSAOs in different species, and sometimes even in different tissues of the same species, can have different substrate-specificities (for a review, see Buffoni, 1983; Lyles, 1994). Second, the nomenclature is based on these functional aspects of the , e.g. inhibitors, co-factors and substrates, without taking into account the genetic relationship between them. The reason for this, in turn, is that genetic information has, until recently, not been available, and still is rather scarce. Since there are no unifying, and yet specific, function-related factors that include all enzymes of the SSAO-family, the result has been that a multitude of names have been used, and most of them have eventually been abandoned. For

8 example, plasma amine oxidase was not a good name since SSAOs are found to a much larger extent in the plasma membrane of adipocytes, smooth muscle cells and in endothelial cells. The name benzylamine oxidase was abandoned since benzylamine is a good substrate also for one of the classical monoamine oxidases, MAO-B. Which leaves us with the name semicarbazide-sensitive amine oxidase, which, unfortunately, is not an optimal name either as described below. As a means of defining the identity of enzymes and ordering them into larger groups, the enzyme classification (EC) system was introduced in the 1950´s. According to this system, all enzymes have been assigned a code number. Based on the traditional function-based way of classifying amine oxidases, they have been ordered into two major groups according to what co- factor they are associated with: One group with flavin-adenine dinucleotide (FAD)-dependent enzymes and one group not containing FAD. The group of FAD-dependent enzymes have, in the EC system been ordered into two separate groups: and B (MAO-A and MAO-B) are categorised as EC 1.4.3.4, and polyamine oxidase (PAO) as EC 1.5.3. The other group is comprised by copper-containing amine oxidases, also referred to as SSAOs. Most of these enzymes are, as the name implies, inhibited by semicarbazide and have a requirement for copper as a co-factor (reviewed in Callingham et al., 1995). With this wide definition, there are a number of amine oxidases included in the term SSAO. Among them we find diamine oxidase (DAO) and (LO) as well as monoamine oxidising enzymes located in plasma or bound to the plasma membrane. All these enzymes are classified as EC 1.4.3.6., except for LO, which is categorised as EC 1.4.3.13 (Siegel, 1979). One problem with the term SSAO is that it nowadays most often is used in a more narrow sense, excluding the semicarbazide-sensitive enzymes DAO and LO. With this more narrow definition, SSAO is used to describe the monoamine oxidising enzymes found in the plasma membrane or circulating in plasma. These enzymes are, apart from being copper-dependent, also

9 dependent on topa quinone (TPQ) as a co-factor (Janes et al., 1990). In other words, in the literature, there is one wide and one more narrow definition of SSAO (Table 1). In order to avoid confusion in this matter, in this thesis I will use SSAO according to the more narrow definition, whereas the term copper- containing amine oxidases will be used referring to the wider definition, including DAO and LO. Before leaving this section on enzyme classification, it ought to be mentioned that there is one more name that is frequently used. SSAO found on the plasma membrane of endothelial cells is sometimes referred to as VAP-1 (vascular adhesion protein 1), since this protein possesses adhesive properties towards leukocytes (see “Physiological role”).

Genetics

The confusion regarding the classification of SSAOs has become somewhat clearer in recent years, since several belonging to this family have been cloned, e.g. three human (Zhang and McIntire, 1996; Imamura et al., 1997; Cronin et al., 1998; Smith et al., 1998), two bovine (Mu et al., 1994; Høgdall et al., 1998), and two murine (Morris et al., 1997; Bono et al., 1998a; Moldes et al., 1999). When the was published, the three human genes that had already been cloned were found to be the only SSAO genes that could be identified, and they were all found on human 17, in 17q21 (reviewed in Jalkanen and Salmi, 2001). One of these genes is a pseudogene (Cronin et al., 1998), whereas the other two code for proteins. These protein- coding genes have been named HPAO (AOC3, human placental amine oxidase, (Zhang and McIntire, 1996)) and RAO (AOC2, retinal amine oxidase, (Imamura et al., 1997)), respectively, and the amino acid-sequence identity between them is 64.4% in the D4 domain (Salminen et al., 1998) (see “Molecular structure and distribution”). The expression-levels of HPAO and RAO differ extensively, HPAO being, by far, the most abundantly expressed. In fact, expression of RAO has only been observed in human retina (Imamura

10 Table 1 Classification of and some characteristics for human amine oxidases

Copper-containing or FAD-dependent amine oxidases semicarbazide-sensitive amine oxidases (SSAOs)

MAO (EC 1.4.3.4) PAO (EC 1.5.3) SSAO (EC 1.4.3.6) DAO (EC 1.4.3.6) LO (EC 1.4.3.13)

Substrates noradrenaline spermine benzylamine putrescine amino groups of spermidine methylamine cadaverine lysine residues adrenaline aminoacetone in collagen and benzylamine elastin

11 Inhibitors chlorgyline MDL 72,527 semicarbazide semicarbazide semicarbazide deprenyl hydralazine carbidopa

Genes MAOA PAO HPAO (hVAP-1) KDAO (PDAO1) LOX MAOB RAO (?) PDAO2

Location mitochondria intracellular cell surface intracellular extracellular plasma

Function degradation of cell growth glucose uptake degradation of formation of neurotransmitters leukocyte adhesion histamine extracellular matrix

FAD: flavin adenine dinucleotide; MAO: monoamine oxidase; PAO: polyamine oxidase; DAO: diamine oxidase; LO: lysyl oxidase; RAO: retinal amine oxidase; Circled SSAO: as the term is defined in this thesis. et al., 1997; Imamura et al., 1998) and because of the very low levels of RAO this enzyme has not been characterised regarding substrate specificity etc. Because of molecular genetics, it is now possible to pin-point the molecular identity of human SSAO. We know that all SSAO found in plasma membranes of various cell-types is encoded by the gene HPAO, and most likely, so is the enzyme found in plasma. As a consequence of this, this thesis concerns the protein that has been encoded by HPAO or its murine orthologue, mVAP-1 (Bono et al., 1998a), and so, in humans the narrow definition of SSAO (above) relates to protein encoded by one single gene. However, it should be noted that since we know very little about RAO, we cannot exclude the possibility that this protein can be excreted into plasma and thus contribute to the plasma SSAO-levels. Genes encoding DAO and LO have also been cloned (Barbry et al., 1990a; Chassande et al., 1994; Trackman et al., 1990; Zhang et al., 1995). DAO has been found to have some sequence similarity to SSAO and RAO (Table 2). In addition to this, the intron/exon structures of these genes are rather similar, suggesting that they may have evolved from a common ancestor gene (Imamura et al., 1998). However, a DAO gene cloned from human has been mapped to human chromosome 7 (Barbry et al., 1990b) and thus is not part of the “SSAO-cluster” in 17q21. LO, on the other hand, is considerably smaller than any of the other copper-containing amine oxidases cloned with a size of 32-kDa in its mature form. The gene encoding this protein is located on human chromosome 5 (Hämäläinen et al., 1991), and it appears that this gene is one member of a larger gene family with a total of five members known today (Kenyon et al., 1993; Mäki and Kivirikko, 2001; Mäki et al., 2001; Saito et al., 1997). These genes are distributed on several different .

12 Table 2 Amino acid and nucleotide (within parentheses) identity, in %, between some mammalian copper-containing amine oxidases

HPAO RAO PDAO1 PDAO2 BSAO rSSAO rDAO mVAP-1

HPAO 100 (6o) c (84)c RAO 64a 100 (62)e PDAO1 41bc 46 a 100 PDAO2 44a 46a 99.8a 100 BSAO 81c 59e 42b 45a 100 rSSAO 83d 38b 78d 100 rDAO 41b 46a 82b 88a 42b 37b 100 mVAP-1 83b 64a 42b 45a 76b 95b 42b 100

HPAO: human placental amine oxidase, RAO: human retinal amine oxidase, PDAO: human placental diamine oxidase, BSAO: bovine serum amine oxidase, rSSAO: rat adipocyte amine oxidase, rDAO: rat diamine oxidase, mVAP-1: mouse vascular adhesion protein 1 (SSAO). a(Salminen et al., 1998). D4 domain only. b(Bono et al., 1998b) c(Zhang and McIntire, 1996) d(Morris et al., 1997). Partial sequence. e(Imamura et al., 1997)

Molecular structure and distribution

The molecular structure of SSAO is sometimes described as mushroom-like, with an N-terminal “stalk”, binding the protein to the plasma membrane, and a large C-terminal “cap”. Or perhaps a more accurate picture would be a field of SSAO-mushrooms protruding from the plasma membrane since estimations have been made suggesting that there are about 14 million SSAO-molecules in the plasma membrane of one single adipocyte (Morris et al., 1997). These “mushrooms” are heavily glycosylated dimers and they consist of two identical subunits with a short N-terminal anchor. As co-factors, each subunit contains one copper ion and an oxidised tyrosine residue, TPQ (Reviewed in Klinman and Mu, 1994), the latter of which interacts directly with the substrate (see “Enzyme reaction, substrates and products”). The tyrosine residue is part of the protein backbone and a lot of interest has been focused upon its conversion to

13 TPQ. It appears that this is a self-processing event only requiring copper and oxygen (Ruggiero et al., 1997). Interestingly, DAO is also dependent on TPQ whereas LO requires lysyltyrosine quinone as a co-factor (Reviewed in Smith- Mungo and Kagan, 1998). The view of human SSAO as a mushroom-shaped molecule is based on sequence alignment with amine oxidases from other species, on the crystal structure of ECAO (an amine oxidase from Escherichia coli (Parsons et al., 1995)), and subsequent structural modelling of the largest domain of the protein, the D4 domain (Salminen et al., 1998). This model shows six potential N-glycosylation sites forming a circle on top of the dimer, whereas no putative sites for O-glycosylation have been found in the D4 domain. The glycosylations of SSAO have caught a lot of interest, especially since they may differ between different tissues, possibly determining the leukocyte-binding property of SSAO (see “physiological role”), and also since estimations have been made, suggesting that more than 10% of the mass of porcine SSAO can be ascribed to surface carbohydrates (Holt et al., 1998). The reason why the other domains were not modelled by Salminen et al, the transmembrane and the extracellular D2 and D3 domains, was that the sequence identity with other copper-containing amine oxidases was low (Salminen et al., 1998). This is interesting in terms of substrate-specificity for these enzymes since the entrance to the , which is deeply buried within the protein, is made up of all three extracellular domains (Wilmot et al., 1997). Another interesting finding is that the three histidines that are binding the copper ion, as well as the tyrosine residue that gives rise to TPQ, are highly conserved in evolution. Altogether, this could imply that the kind of reaction catalysed by copper-containing amine oxidases, and therefore the structure of the active site, is essential in all living organisms, but that the substrate specificity is secondary (Table 3).

14 Table 3 near the TPQ- precursor, tyrosine (bold), in some copper-containing amine oxidases

Gene Amino acids

HPAO NYDYab RAO NYDYbc PDAO1 NYDYb PDAO2 NYDYb BSAO NYDYabcd rDAO NYDYbc mVAP-1 NYDYb LNSAO NYDNd YPAO NYEYcd ECMAO NYDYc AGHAO NYDYc AGPAO NYDYc

Abbreviations: N: Asparagine, Y: Tyrosine, D: Aspartic acid, E: Glutamic acid, HPAO: human placental amine oxidase, RAO: human retinal amine oxidase, PDAO: human placental diamine oxidase, BSAO: bovine serum amine oxidase, rDAO: rat diamine oxidase, mVAP-1: mouse vascular adhesion protein 1 (SSAO), LNSAO: lentil seedlings amine oxidase, YPAO: yeast Hanensula polymorpha peroxisomal amine oxidase, ECMAO: Escherichia coli monoamine oxidase, AGHAO: Arthrobactor globiformis histamine oxidase, AGPAO: Arthrobactor globiformis phenylethylamine oxidase. Sequence alignments presented in: a(Zhang and McIntire, 1996), b(Salminen et al., 1998), c(Imamura et al., 1997), d(Mu et al., 1994).

SSAO is mainly located in the plasma membrane of adipose-, smooth muscle- and endothelial cells, in that order of magnitude. In fact, it has been estimated to make up about 2.3% of the total amount of protein found in the plasma membrane of adipocytes (Morris et al., 1997). The origin of plasma SSAO has been a matter of speculation, but evidence is now accumulating suggesting that, at least in humans, it is a cleavage product of the transmembrane form (Stolen et al. 2001, Göktürk et al., unpublished observations). Possibly, it is a result of proteolytic cleavage of the N-terminal sequence, but this has not yet been established.

15 Enzyme reaction, substrates and products

As mentioned previously, there is a large diversity regarding the substrate- specificity for SSAO enzymes. Nevertheless, the basic reaction is the same: They all catalyse the deamination of an amine, forming an aldehyde, and ammonia. In general, the substrates are aromatic or aliphatic primary amines. The best endogenous substrates for human SSAO known today are methylamine and aminoacetone, although neither of these two monoamines is a particularly good substrate (Table 4). The corresponding aldehydes produced are formaldehyde and methylglyoxal (2-oxopropanal), respectively, two highly reactive molecules. It should be noted, however, that SSAO in pig as well as in human dental pulp recognises 5-hydroxytryptamine (5-HT) as a substrate (Norqvist et al., 1981; O'Sullivan et al., 2002). Another feature specific for SSAO in dental pulp, is that the enzyme has been observed in nerve fibres of this tissue (O'Sullivan et al., 2002).

Table 4 Km (µM) values for amines serving as substrates for human SSAO

Source Methylamine Aminoacetone 5-HT Benzylamine

Umbilical artery 832a 92b, 126c - 222a, 155d Plasma 516a N/A - 225a, 279d Dental pulp N/A N/A 318e 254e a(Lyles et al., 1990); b(Lyles and Chalmers, 1992); c(Deng and Yu, 1999); d(Yu et al., 1994); e(O'Sullivan et al., 2002). N/A= Not available. – Not a substrate.

Aminoacetone can be ingested, but it is also formed in vivo from the condensation of acetyl coenzyme A and glycine, or by the oxidation of L- threonine (von Studnitz, 1967) (Fig. 1). Methylamine can be derived from

16 several metabolic pathways, e.g. it is formed when adrenaline, sarcosine or creatinine are degraded (Schayer et al., 1952; Davis and De Ropp, 1961; Yu et al., 1997; Mitchell and Zhang, 2001)(Fig. 2). In addition to these endogenous sources, methylamine can also be produced by gut-living bacteria, it can be ingested with the food (e.g. fish) or inhaled with cigarette smoke (US Department of Health and Human Services, 1982, Mitchell and Zhang 2001).

17 Apart from these endogenous amines, SSAO also recognises, e.g. benzylamine and allylamine as substrates. Of particular interest is allylamine, which can form the highly reactive substance acrolein (Boor and Hysmith, 1987), which is known to be a cardiovascular toxin. The reaction catalysed by SSAO is a so-called ping-pong reaction and can be divided into two separate half-reactions: One reductive and one oxidative (Fig. 3). In the first half-reaction the amine group of the substrate interacts with the TPQ in the active site, reducing the enzyme and producing a Schiff base between the substrate and the TPQ co-factor. Hydrolysis then occurs releasing an aldehyde as a first product in the reaction. In the second half-reaction, the reduced TPQ is re-oxidised in a reaction that is dependent upon copper and molecular oxygen, under the production of hydrogen peroxide and ammonia. All of these products are toxic in high concentrations and will be discussed further under “Toxicity”.

18 Reductive half-reaction

E-CHO + R-CH2-NH2 E-CH2-NH2 + R-CHO

Oxidative half-reaction

E-CH2-NH2 + O2 + H2O E-CHO + NH3 +H2O2

Figure 3. The two half-reactions catalysed by SSAO enzymes.

The products in the SSAO-catalysed reactions are highly reactive and, as would be expected, they are further metabolised. Hydrogen peroxide can be degraded by glutathione peroxidase and by catalase, whereas excess ammonium ion is converted to urea, which is excreted. The aldehyde methylglyoxal is degraded by the glyoxalase system or by methylglyoxal dehydrogenase (Reveiwed in Callingham et al., 1995), forming pyruvate that can enter the citric acid cycle. Formaldehyde is further metabolised by formaldehyde dehydrogenase, forming formic acid (Reviewed in Heck et al., 1990).

Physiological role

Is SSAO of physiological importance? One would assume that it has got important functions considering its widespread occurrence, but still, the answer to this question is not quite clear. The search for plausible physiological functions for these enzymes has been confounded by the fact that the substrate specificity differs a lot even between the mammalian species, and that no really good endogenous substrates have been found for human SSAO (Table 4). Over the years it has been speculated that the enzyme may be involved in a “first line of defence” against potentially harmful monoamines, inhaled or ingested. However, since the amines that head the list of substrates for SSAO give rise to products that are more cytotoxic than the parent compounds, the physiological value of such a defence has been questioned. Nevertheless, it is still possible

19 that SSAO may provide some protection against other monoamines, since it is somewhat reactive towards substrates more readily deaminated by the classical MAOs, e.g. 2-phenylethylamine, tyramine and dopamine. In fact, the SSAO activity has been observed to be transiently increased in rat heart as a result of MAO-inhibition, suggesting that SSAO may, in part, provide some compensation for dysfunctional MAO-activity (Fitzgerald and Tipton, 2002). However, it should be noted that these “MAO-substrates” are very weak substrates for human SSAO (reviewed in Lyles, 1994), and that a response similar to that observed in rat therefore may not be evident in man. In the last few years, advances have been made in this area and despite the toxicity, focus is now turning more to one of the products in the reaction. It appears that the formation of hydrogen peroxide is pivotal for at least one of the functions that are now being ascribed to the enzyme. Hydrogen peroxide is toxic at high concentrations, but at lower concentrations it has got important functions as a signal-transducing molecule and it may well be that we will find more important functions for SSAO relating to its ability of forming this product. When produced by SSAO, it has been observed to be important for the translocation of the glucose-transporters Glut1 and Glut4 to the plasma membrane, in an -mimicking manner (El Hadri et al., 2002; Marti et al., 1998). In this way, SSAO-mediated formation of hydrogen peroxide has the ability of promoting uptake of glucose into the cells. Hydrogen peroxide is also known to affect the function of, for example, the transcription factor NFκB. Via this mechanism, SSAO could perhaps affect gene-expression, and in this respect, another finding becomes very interesting. The expression levels of SSAO is increased more than 100-fold as adipocytes differentiate (Moldes et al., 1999) and an appealing thought would be that SSAO is involved in this differentiation. One function that is exclusive for endothelial SSAO, with respect to SSAO found on other cell-types, is the ability to mediate binding of leukocytes (Jaakkola et al., 1999). This is a function that became apparent to the researchers in the SSAO-field only in 1998, when a Finnish group of

20 researchers cloned a gene coding for the protein they called vascular adhesion protein 1 (VAP-1) (Smith et al., 1998). Most likely, they were as surprised as we were, when they found that their protein had already been cloned under the name human placental amine oxidase (HPAO) (Zhang and McIntire, 1996). In this way, two fields of research converged into one and it became obvious that SSAO found on endothelial cells plays a role in the recirculation of lymphocytes and also in attracting leukocytes to sites of inflammation. Such guiding of leukocytes is a result of a cascade of sequential events, sometimes referred to as “homing” (Fig. 4). The leukocytes first tether to the endothelium, then start to roll. If they are activated by appropriate signals, they attach to the endothelium and start to transmigrate into the inflamed tissue, a process sometimes referred to as diapedesis. In this cascade of events, SSAO appears to be important in the rolling phase as well as in the transmigration step. Among mononuclear cells, it specifically directs CD8-positive T killer cells and natural killer cells, but it also supports adhesion of granulocytes (Salmi et al., 1997; Tohka et al., 2001; Lalor et al., 2002). The exact mechanism behind this has not yet been established, but it appears that the enzyme activity is crucial for the adhesion to take place (Salmi et al., 2001). Altogether, SSAO is now emerging as a family of enzymes with important cell signalling functions (reviewed in Jalkanen and Salmi, 2001) and in conclusion, most likely a lot is still to be discovered in this field. One example of a question without an answer concerns the circulating SSAO. We still do not know if plasma SSAO has got important tasks to fulfil. Perhaps it is merely a result of shedding of protein from the endothelium, but it could also be that it is of importance in its own right, for example, in activating leukocytes.

21 BLOOD STREAM LEUKOCYTE

Tethering Rolling Activation Arrest

ENDOTHELIUM Diapedesis

INFLAMMATION

Figure 4. Homing is a multi-step process in which several adhesion molecules are involved as a means of directing leukocytes. In this process, leukocytes in the blood first start to tether to the endothelium, often as a result of interactions between selectins and carbohydrates, and then start to roll. If the leukocytes are activated, they will arrest, and transmigrate through the vessel wall. SSAO (VAP-1) appears to be involved in the rolling phase as well as in the transmigration step of this process.

Toxicity

There have been several reports describing the toxicity of the products in SSAO-catalysed reactions (Boor et al., 1990; Yu and Zuo, 1993; Callingham et al., 1995). Indeed, the list of products may sound alarming: Formaldehyde, methylglyoxal, hydrogen peroxide, ammonia and possibly even acrolein from exogenous sources of allylamine. It is well-known that hydrogen peroxide can be transformed to the highly reactive hydroxyl radical and thereby can contribute to . Ammonia in high concentrations is neurotoxic due to its excitatory actions on glutamate-receptors in the brain (reviewed in Albrecht, 1998) and acrolein causes severe cardiovascular lesions (Boor and Hysmith, 1987). But the main focus regarding the possibly injurious effects of SSAO has been on the aldehydes produced from endogenous substrates, methylglyoxal and formaldehyde.

22 Both methylglyoxal and formaldehyde are well-known mutagens and have the capacity of cross-linking DNA and RNA. In addition to this, they have the capacity of forming adducts with proteins: Formaldehyde has been reported to bind irreversibly to lysine-residues of proteins, whereas methylglyoxal binds irreversibly to arginine-residues (for a review, see Heck et al., 1990; Thornalley et al., 1995). Consequently, these aldehydes can damage both DNA and proteins. In fact, methylamine has been shown to be cytotoxic in vitro in the presence of SSAO, suggesting that the cytotoxicity may be a result of the formaldehyde-production (Yu and Zuo, 1993). Additionally, methylglyoxal is very potent in forming early glycation products, and spontaneous in vitro glycation is enhanced in the presence of formaldehyde (Yu and Zuo, 1997). In other words, both these aldehydes may play important roles in the formation of advanced glycation end-products, AGEs (see “Diabetes mellitus”). The possibly harmful effects of SSAO-mediated production of these substances may sound compelling, but it has to be looked upon with some perspective. For example, it should be noted that formation of methylglyoxal is not unique for SSAO since, for example, it can also be formed from acetone and triosephosphate, and we do not know the proportion of the SSAO- contribution. It could be very minor compared to the other sources. The same can be argued for formaldehyde, ammonia and hydrogen peroxide. There are numerous sources of these substances physiologically and we can only speculate about the relative amount produced by SSAO. With this perspective, the contribution from SSAO activity may, or may not, be of relevance. No matter which is the case regarding the total exposure to these substances, it is still possible that the local effects of high SSAO activity are detrimental. It is also possible that the input from SSAO activity within the normal range is minor in relation to other sources, but that it is of significance only after prolonged exposure to increased activity.

23 Diabetes mellitus

There appears to be a connection between SSAO and diabetes, partly because of the fact that the enzyme activity is increased as a result of the onset of the disease (Hayes and Clarke, 1990; Elliott et al., 1991; Boomsma et al., 1999; Article IV), and partly because of the insulin-mimicking effect of hydrogen peroxide (Marti et al., 1998; El Hadri et al., 2002). Additionally, the aldehyde- products in the SSAO catalysed reactions may contribute to the formation of AGEs, which have been suggested to be implicated in the development of vascular complications in diabetes (below). Diabetes mellitus is a group of disorders characterised by elevated blood glucose concentrations, and they are commonly divided into two groups. Type 1, or insulin dependent diabetes mellitus (IDDM), is the classical form of diabetes and these patients cannot survive without insulin treatment. In most cases, this is a disease of autoimmune background in which the insulin- producing β-cells in the pancreas are destroyed. In type 2, or non-insulin dependent diabetes mellitus (NIDDM), the insulin released from the β-cells is not sufficient, or there may be a decreased insulin-sensitivity in the tissues. In order to control the blood glucose levels, these patients are kept on a restricted diet and they are sometimes treated with oral hypoglycaemic agents (OHA). They are treated with insulin only if diet and OHA-treatment is not sufficient for controlling the blood glucose concentrations. More than 90% of all diabetes-cases in the world are classified as type 2, and the risk of developing this type of diabetes increases with age, high glucose levels, and obesity. Additionally, there appears to be rather strong genetic components determining the risk of developing type 2 diabetes. Due to the fact that people are living longer, and due to the connection with obesity, the prevalence of type 2 diabetes in the world is growing (reviewed in Zimmet et al., 2001) With progression of diabetes, changes in the blood vessels often occur, leading to complications such as retinopathy, nephropathy, and neuropathy. As a consequence of the micro- and macrovascular pathology, diabetes is a

24 common cause for blindness (Kohner et al., 1991) and end-stage renal disease, it is risk-factor for stroke, myocardial infarction, limb amputation, and increased mortality from cardiovascular disorders. The complications can be delayed by thorough glycaemic control, but there is no existing cure. The underlying mechanisms controlling the development of vascular damage are still unclear, but there are a number of hypotheses. One of these hypotheses is that there is an increased formation of AGEs. The initial event in the formation of irreversible AGEs is the formation of a Schiff base between a sugar aldehyde or ketone and a free amino group of a protein or nucleic acid, forming slowly reversible Amadori products (Bucala et al., 1984; De Bellis and Horowitz, 1987). Originally, this was thought to be a result of non-enzymatic reactions between extracellular proteins and glucose. However, since Schiff base formation is directly proportional to the percentage of sugar in the open chain form, and since only 0.002% of all glucose exists in this form, this particular sugar has the slowest rate of Schiff base formation of any sugar found in cells (Bunn and Higgins, 1981). Instead, it would appear that dicarbonyls such as 3-deoxyglucosone, glyoxal and methylglyoxal are much more potent in forming these early glycation products (Reviewed in Brownlee, 2001), which eventually, after prolonged exposure to high glucose levels, are transformed into irreversible AGEs. Since AGEs are irreversibly bound to macromolecules, they accumulate in the diabetic tissues, e.g. there is a threefold increase of AGEs in diabetic arterial wall collagen (Makita et al., 1991). In this way, AGEs can cause changes in extracellular matrix components that will affect their function, possibly by altering their normal interactions with other cells. In diabetic vessels this may lead to an overproduction of matrix components as well as an abnormal adhesion of immune cells to the endothelium. In this way, there may be a causal relationship between AGE formation and the development of vascular damage in diabetes.

25 SSAO in diabetes

Although the “proof of concept” is still lacking, the circumstantial evidence is rather convincing that SSAO is involved in the development of vascular complications in diabetes. Apart from the fact that the activity is increased, the levels of aminoacetone (Jerzykowski et al., 1968) and methylglyoxal (McLellan et al., 1992) are also increased, as are the AGE-levels. Additionally, the oxidative stress in diabetes is known to be high (for review, see Brownlee, 2001). It is known that the SSAO activity is increased in the tissues as a result of the onset of diabetes, and consequently, one would expect an increased production of toxic metabolites. This, in turn, could therefore lead to an increased formation of AGEs and oxidative stress as discussed above. In addition to these direct effects, there is also a possibility that endothelial SSAO, as a consequence of its leukocyte-adhesive properties, could contribute to vascular complications, since it is known that diabetic microangiopathy is associated with capillary occlusion by leukocytes (Schröder et al., 1991). Altogether, this has led many investigators to suggest a possibility of reducing or preventing the development of diabetes-associated vascular complications by use of specific SSAO inhibitors (Yu and Zuo, 1993; Ekblom, 1998). Due to the fact that there are SSAO-inhibitory drugs in clinical use, we know that it is possible to be standing on a long-term treatment with such a drug. For example, two of the drugs used for treating Parkinson´s disease, carbidopa and benserazide, besides inhibiting the dopa-decarboxylase, also inhibit SSAO activity. Also the drug hydralazine, which was previously used to treat high blood pressure, is a potent SSAO inhibitor. However, it is more or less impossible to try to speculate whether an inhibitor would actually have pharmacological value, and whether lowered levels of enzyme activity would be accompanied with unwanted side-effects specific for diabetic patients. It may be argued that there could be a reason for the increased activity seen in relation to diabetes, and the possibility has to be taken into account that a

26 reversal of this effect is more injurious than the effects of high SSAO activity. For example, SSAO´s ability of affecting glucose-uptake via Glut1 and Glut4 may be necessary in diabetes. In connection with the development of new pharmacological treatment strategies, the role SSAO plays has to be much better understood. For example, it is not known whether the increased activity is a direct cause for the vascular damages, or is simply to be seen as a risk-marker. We know that it is not a marker for vascular damage, since the activity is increased as a result of the onset of diabetes, long before any vascular complications arise (Hayes and Clarke, 1990; Elliott et al., 1991; Boomsma et al., 1999). Nevertheless, it may still be a marker for other events taking place early in the course of the disease, events that eventually may be the direct cause for the vascular complications.

PRESENT INVESTIGATION

Aims of the thesis

The work that we have conducted in the field of SSAO has been focused on two major issues, namely the regulation of the enzyme activity and its possible involvement in the development of vascular damage. We wanted to get closer to understanding why the activity is high in certain diseases, such as diabetes. In order to explore these issues we have used several approaches using biochemical and molecular methodologies as well as clinical studies, and the main model for our investigations has been diabetes.

Article I

The aim of the work presented in article I, was to examine how metabolites of methylamine are distributed in the tissues, and how long time it would take to regain normal SSAO activity after irreversible inhibition of the enzyme. We

27 wanted to get an overview over the tissues exposed to the possibly toxic effects of high SSAO activity, and thus also get an indication as to whether the reaction could be involved in the development of vasculopathies. The question regarding recovery rate of the enzyme is of importance as a starting point for understanding the mechanisms controlling the enzyme activity. To approach these issues, we treated mice with intraperitoneal (i.p.) injections of 14C-methylamine and studied the distribution of radioactivity in mice with normal SSAO activity, and compared this with the distribution found in mice that had been pre-treated with an SSAO-inhibitor, hydralazine. In this way, we could follow the reaction product carrying the 14C-label, and we could also see what effect an enzyme-inhibitor would have on the production. We did this by using an autoradiographic method, looking at whole-body tissue sections of the animals. In short, two doses of saline or hydralazine hydrochloride were given i.p. 24 hours apart. Twenty-four hours after the last injection of hydralazine or saline, the mice were given an i.p. injection with 14C-methylamine, and 24 hours after that, the mice were sacrificed and frozen at -20°C. The animals that had been pre-treated with hydralazine are referred to as ‘day 1’-mice whereas the saline-treated animals are controls. In order to study the recovery of SSAO activity after inhibiting the enzyme irreversibly, we administered the 14C-labelled substrate to four additional groups of animals two, four, seven, or fourteen days after the last injection with hydralazine. Just as the controls and the ‘day 1’-mice, these animals were sacrificed 24 hours after the injection of substrate. The mice were then frozen in cubes with carboxymethyl cellulose (CMC) and 20 µm thin sagittal tissue sections were cut in a cryo-microtome. The sections were collected on adhesive tape and dried at -20°C for at least 24 hours. The tapes were thereafter exposed on 14C- sensitive film at -20°C for seven weeks, before development. The effect of the hydralazine-treatment was striking since it led to an almost complete reduction of incorporated radioactivity (Table 5). We therefore concluded that the radioactive residues observed in the control- animals were the result of incorporated metabolite, most likely 14C-

28 Table 5 Optical density in controls, and relative inhibition of incorporated 14C-methylamine-metabolite after treatment with hydralazine.

Tissue Control OD % of control, day 1

Small intestine 74.6 13.4 Spleen 62.5 12.3 Adipose tissue 62.2 5.0 Bone marrow 55.4 11.0 Salivary gland 48.1 13.4 Kidney 33.5 16.1 Liver 32.5 22.2 Skeletal muscle 19.6 11.2 Lung 10.4 28.8 Brain 7.9 37.6

OD: Optical density (units).

formaldehyde. We estimated the optical density (OD) for the different organs in each animal using the Macintosh software NIH image (BrainImage Pascal 2.3.3). The tissues that contained the highest levels were small intestine, spleen and adipose tissue, but also bone marrow and salivary gland displayed high levels. The levels in kidney and liver were intermediate, whereas the central nervous system was practically devoid of metabolite. Also lung and skeletal musculature contained very low levels (Fig. 5). By injecting 14C-methylamine at different time-points after SSAO inhibition with hydralazine, we were able to study the recovery rate of SSAO activity in different tissues, presumably reflecting de novo synthesis of the protein. We found that adipose tissue had a normalisation-time that was about twice as long as in any other tissue examined (Fig. 6). In order to confirm that the SSAO activity was indeed inhibited by hydralazine, and to get an indication as to whether the OD in the autoradiographs directly corresponded to the enzyme activity, we quantified the SSAO activity in some of the tissues. We use a radiochemical assay for

29 determining SSAO activity. The principle for this method is that we pre- incubate the samples for 20 minutes in room temperature with the MAO- inhibitors chlorgyline and L-deprenyl, and blank samples are also pre-treated with hydralazine. We then administer a certain amount of 14C-labelled enzyme- substrate (benzylamine) to the tissue homogenates, serum or plasma samples, incubate the mixture in a 37°C water-bath for 20 minutes, and then stop the reaction by addition of HCl. The 14C-labelled benzaldehyde product is

30 extracted with toluene: ethylacetate 1:1 and quantified by scintillation in a Packard Tri-Carb Liquid Scintillation Analyzer, model 1900 CA®. The results showed us that the inhibition indeed was efficient in the hydralazine-treated animals. We also found that there was no direct connection between SSAO activity and OD in the autoradiographs. This could reflect e.g. variations in distribution of substrate and/or product between different tissues, or variations in the occurrence of formaldehyde dehydrogenase.

Article II

In this study, our intention was to repeat what we did in article I, although with the substrate aminoacetone labelled with 14C. The reason was the same as in the former study, to see in what tissues a possibly toxic effect, resulting from SSAO-mediated aldehyde-formation, would be expected. We wanted to see if the distribution patterns would be similar with the two substrates, and also to compare this with the distribution of SSAO activity. An additional question we wanted to answer in this study was how long-lived the radioactive deposits were in the tissues. The methodology in this study was the same as described above for methylamine, with one major difference. In this case, we did not study the recovery of SSAO activity after irreversible inhibition of the enzyme. Instead, we wanted to evaluate the longevity of the 14C-labelled products in the reaction. Therefore, in this study, we administered the 14C-aminoacetone to all animals 24 hours after the last injection with hydralazine, but we varied the time before sacrificing them. The mice were sacrificed one, four or eight days after the injection with 14C-aminoacetone. This time, the strongest degree of incorporated radioactive substance was in adipose tissue and Harderian´s gland. The central nervous system was also this time devoid of radioactive residues, and this is in agreement with the fact that the SSAO activity in brain is very low. All other tissues displayed a very homogenous degree of incorporation that could be described as intermediate.

31 Compared to study I, the most striking difference was that the tissues small intestine, spleen, salivary gland and bone marrow had much lower levels of radioactive residues. In this study, these levels were about the same as the levels in liver and kidney (Fig. 7).

A problem for the interpretation of the results in this study was that the distribution of radioactive residues was the same, regardless of whether we had, or had not, pre-treated the animals with the SSAO-inhibitor, hydralazine. In other words, with this result we could not exclude the possibility that what we saw in the autoradiographs was aminoacetone itself rather than a product from the SSAO-catalysed reaction. However, we found it less likely to be aminoacetone since there are indications suggesting that this amine may be relatively short-lived in the tissues (Marver et al., 1966), and our results indicated that there were radioactive residues still remaining in the tissues after eight days. Additionally, there are studies suggesting that aminoacetone can be degraded non-enzymatically in vitro (Dutra et al., 2001; Hiraku et al., 1999). We therefore came to the conclusion that there is a possibility that our findings

32 support these results from in vitro assays, and that aminoacetone could be degraded non-enzymatically also in vivo. In conclusion, although unable to provide conclusive evidence as to whether SSAO-inhibition would result in reduced incorporation of methylglyoxal, this study did not support the idea.

Article III

Article III is a follow-up of a study that had been performed almost three years earlier. In the original study, 65 patients with type 2-diabetes were studied and it was found that the SSAO activity was high in these subjects, as compared to healthy controls, and that it was even higher in presence of retinopathies (Garpenstrand et al., 1999). In this study, 34 of the patients from the original study participated, and our aim was to examine whether the enzyme activity or the retinopathy-status had changed significantly over time. We also examined the levels of glycosylated haemoglobin (HbA1c) and the concentrations of methylamine in urine. The patients that were included in this study visited a primary health care centre for a routine check-up of their retina status. Having received written information, they all gave informed consent to participate in the study. This meant that they agreed to donate blood for analysis of plasma SSAO activity as well as for HbA1c, and a sample of urine for analysis of methylamine concentration. The method for determining SSAO activity was the same as described above (article I). Analyses of HbA1c were performed at Huddinge University Hospital, using ion exchange chromatography, and the urinary concentrations of methylamine were measured by the use of a fluorometric HPLC method (Yu and Dyck, 1998). The degree of retinopathy was determined by fundus photography, which is considered to be a very sensitive method for detecting retinopathy. Briefly, the pupils of the patient are dilated with tropicamide before fundus photographs are taken, and the slides are

33 thereafter graded according to criteria set up by the ETDRS (Early Treatment Diabetic Retinopathy Study) (Kohner and Porta, 1991). Once again, we could establish a connection between retinopathy and elevated SSAO activity. Since the time that had passed between the two studies was relatively short, we could not draw any conclusions regarding the development of retinopathy, but we could see that the enzyme activity was rather stable between the two occasions (Fig. 8). SSAO activity is relatively stable in healthy individuals and we could now draw the conclusion that the same also seems to be the case in these diabetic patients controlled for glycaemia. We also found that urinary levels of methylamine were lower among the group of patients with retinopathy, further enhancing the connection between methylamine deamination, SSAO activity and retinopathy.

Additionally, we found that HbA1c is positively correlated with plasma SSAO activity, possibly implying that glucose levels are involved in the activation of the enzyme.

I

I 30

I

e

l

c

i

t

r

A 20

,

p

u

-

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o 10

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l

o F 0 0 102030405060

Baseline, 1996

Figure 8. Plasma activity of SSAO in 34 type 2 diabetes patients in 1999, versus the activity reported in the baseline study performed in 1996.

34 Article IV

Very little is known about how the SSAO activity is regulated, and this issue was the main focus of article IV. It has been known for quite some time that the activity is increased when diabetes is induced in animals (Hayes and Clarke, 1990; Elliott et al., 1991), but there have been no reports about what mechanisms are responsible for the increased activity. From article I and article III, we had found that there was a possibility that the activity was differently regulated in different tissues, that there was a connection between high blood glucose levels and increased enzyme activity, and that the activity was quite stable also in diabetes. In this study, we examined how the expression of the SSAO-gene was affected when the enzyme activity is increased after induction of diabetes in mice. Diabetes was induced in the mice by a single intravenous (i.v.) injection of alloxan, and the mice were sacrificed 24 hours, or seven days, later. Blood glucose levels were determined and were considered to be in the diabetic range if they exceeded 12 mmol/l. The alloxan-treated animals that had not reached this level were excluded from the study. Serum and tissue samples were collected and analysed for SSAO activity by use of the radiometric assay described above (article I). Additionally, total RNA was prepared from lung and adipose tissues. For quantification of SSAO-mRNA, we developed a protocol for real-time PCR. Briefly, we synthesised cDNA from the RNA- samples, and this cDNA was used as a template in a PCR-reaction to which we had added a fluorophore for detection of PCR-product, in real time. The fluorophore (FAM) and a quencher (Tamra), that quenches the fluorescence that is emitted from FAM, are bound to a TaqMan-probe that specifically recognises the SSAO-PCR-product (Fig. 9). The fluorescence from FAM is emitted, and thereby detected, only if the TaqMan-probe is cleaved and the quencher in that way is separated from the fluorophore. This happens only if the probe is tightly bound to the template DNA, so that the probe is broken when the Taq-polymerase, used to elongate a new strand of DNA in the PCR-

35 A

FAM Tamra primer 5´ 3´ template cDNA

Tamra B FAM primer Taq template cDNA

Figure 9. The TaqMan probe binds specifically to the SSAO gene on the template. The primers for the PCR have been designed to lie very close to the probe, thereby giving rise to a short PCR-product. The probe should have a Tm that is approximately 10oC higher than the Tm of the primers, so that it is firmly attached when the primers anneal, and when elongation occurs (A). When the Taq-polymerase elongates the PCR-product, the probe will break up if it has annealed to the right sequence on the template. In this way, the quencher will be separated from the fluorophore, and fluorescence will be emitted when the reaction is irradiated with UV-light (B).

reaction, tries to get by the probe. This is considered to be a very sensitive method for quantifying mRNA-levels. We found that when the activity in adipose tissue is increased, the SSAO-mRNA levels are decreased (Fig. 10). We could see this already 24 hours after the diabetes-induction, as the blood glucose levels started to rise. We also found that this effect was still remaining after seven days. In lung tissue, the activity increased after 24 hours, but had returned to normal after seven days. In this tissue, however, we could not see that an increased activity was connected with decreased mRNA levels. We also found that the SSAO activity in serum was increased seven days after the alloxan-treatment, and that this activity was positively correlated with blood glucose levels (Fig. 11).

36 The main conclusion drawn from this study was that the SSAO activity was not increased as a result of increased gene transcription. Instead, since we found lowered mRNA-levels in adipose tissue, we conclude that something happening early in the course of the disease increases the activity post-

37 transcriptionally. Most likely, it is affecting the protein post-translationally, perhaps by affecting the glycosylation pattern or the cofactors associated with the enzyme. We speculate that this increased enzyme activity, or something happening in parallel with it, affects the gene-expression negatively in adipose tissue, i.e. that there is a negative feedback loop. We did not find proof for such a negative feedback loop in lung tissue, either reflecting that the sample was too small to detect a down-regulation of mRNA as a result of diabetes, or that the activity in this tissue is, in fact, differently regulated. The fact that the enzyme activity in serum was positively correlated with blood glucose levels seven days after the induction of diabetes may imply that glucose could be important either in activating the enzyme, or in regulating its release from the blood vessel wall.

38 CONCLUSIONS

The main conclusions from this thesis are:

• Pharmacological treatment with an SSAO-inhibitor would potently diminish the deamination of methylamine, with a subsequent reduction of metabolite incorporated in the tissues. • We raise the possibility that the SSAO activity may be differently regulated in different tissues. • We did not find support for the hypothesis that an SSAO-inhibitor would decrease the deamination of aminoacetone and thus affect the formation of methylglyoxal. • SSAO activity appears to be stable over time in type 2 diabetes controlled for glycaemia. • Retinopathy in diabetes is associated with increased SSAO activity and decreased urinary levels of methylamine, further strengthening the relationship between enzyme activity and vascular complications.

• Plasma or serum SSAO activity is positively correlated with HbA1c in diabetic patients, as well as with blood glucose in alloxan-treated mice. This may suggest a role for blood glucose either in activating the enzyme activity, or in promoting its release into the blood stream. • Increased SSAO activity in diabetes is not a result of increased SSAO gene transcription. • We raise the possibility of a negative feedback on the SSAO gene expression in adipose tissue, counteracting increased SSAO activity.

39 GENERAL DISCUSSION AND FUTURE PERSPECTIVES

Possible benefits of SSAO

We are now at the beginning of understanding at least some of the functions that can be ascribed to SSAO. But still, the lack of specific substrates for the enzyme is puzzling. One possible explanation for this lack of good substrates could be that the importance of SSAO enzymes does not lie in their ability of deaminating certain amines, but rather in their ability of forming certain products, as suggested by Callingham and Barrand already in 1987 (Callingham and Barrand, 1987). For example, hydrogen peroxide is becoming renowned as a signal-transducing molecule, and it could well be that an important function for SSAO lies in its ability of directing the production of this particular molecule. Perhaps this is the reason for the large diversity regarding substrate-specificity among SSAOs? Perhaps SSAO can use just about any primary amine available simply in order to produce hydrogen peroxide? Since there are indications suggesting that the production of hydrogen peroxide could be a key issue in explaining the mere existence of SSAO, one could speculate that the aldehydes and the ammonia are by-products in the reaction. It could be that these substances can be accepted at low concentrations because the benefits of producing hydrogen peroxide outweigh the disadvantages of such by-products. However, if this is the case, there could be a fine line determining whether the production of these metabolites is beneficial or detrimental. As a consequence of this, it would be of great importance that the enzyme activity is under rigorous control. A negative feedback-mechanism, such as suggested in article IV, would contribute to

40 keeping the enzyme activity at a stable level. Nevertheless, the activity is affected in some physiological and pathological conditions, and we still know very little about the factors causing this.

Possible regulatory mechanisms

Hayes et al. showed that the increased activity in diabetes is a result of increased Vmax rather than a change in Km (Hayes and Clarke, 1990). This, together with our findings presented in article IV, would imply that despite the fact that the de novo production most likely is reduced, since the mRNA-levels were decreased, the number of catalytically active protein-molecules is increased. In my view, the first place to look for an answer to the question of how the enzyme activity is increased in diabetes would therefore be at the post- translational level. Perhaps under normal conditions there is a rather constant SSAO gene-expression giving rise to a pool of protein, some of which is catalytically inactive. Such a pool of inactive enzyme could be of importance due to its potential of giving a fast response to, for example, increased concentrations of glucose. If this is true, biochemical changes as a consequence of diabetes could lead to sustained high enzyme activity by causing a shift in the ratio between active and non-active SSAO. In this respect, the conversion of tyrosine to TPQ is highly interesting, since this could be the activating factor. Another possible explanation for the increased enzyme activity in diabetes could be that the degradation of the protein is slower. It is well-known that glycosylations affect the tertiary structure of proteins. Prions, for example, are less degraded in their pathogenic conformation and it has been speculated that this is a result of an altered glycosylation pattern (Rudd et al., 2001). Since the SSAO protein is heavily glycosylated, it could be that the biochemical changes that are associated with the diabetic state somehow affect these glycosylations, and thereby shield the protein. In this way, perhaps SSAO

41 could be protected when proteins are degraded for energy-production in diabetes.

Investigations ahead

As a concluding remark, this thesis has provided an approximate equal number of questions and answers to the field of SSAO. For example, is the gene expression controlled by the activity of the enzyme it is encoding? Could such a negative feedback be mediated by a product in the reaction? Could it be that glucose is involved in the activation of the enzyme since there is a correlation between blood glucose and plasma SSAO activity? Or is the glucose level involved in the release of SSAO from the vessel wall?

Continuously, new questions follow and the story still goes on.

42 ACKNOWLEDGEMENTS

Först av allt skulle jag vilja rikta ett stort, allmänt tack till alla nuvarande och tidigare kollegor på institutionen. Tack för all hjälp och för att ni alla bidragit till den trevliga atmosfären!

Jag skulle särskilt vilja uttrycka min tacksamhet till:

Min handledare Lars Oreland. Tack för att du givit mig förtroendet att doktorera hos dig, och för att du är noga med att man ska fika också! Jag vill även tacka dig för att du vågar vara den typ av handledare som ger sina doktorander förtroendet att arbeta självständigt, och som bistår med hjälp när det verkligen behövs.

Jonas Ekblom som var min entusiastiske handledare på lab när jag började. Lycka till i Amerikat!

Alla i gruppen! Allrahelst Camilla. Vilket samarbete! Mattias, Cecilia, Håkan och Sigrid. Så många kör-talanger på ett och samma lab! Iggebagge låter helt enkelt inte bra utan er. Tack också till Elisabeth och Jonathan och andra som jobbat någon period i vår grupp under min doktorand-tid.

Alla mina medförfattare. Jag hade inte kunnat göra det här utan er.

Dan-lab. Tack för lånet. Jag tror att jag har lämnat tillbaka allt. Ett särskilt tack till Dan Larhammar själv för att du såg till att förse mig med ett bra ex-jobb och en medföljande trevlig atmosfär.

Alla i Johan Stjernschantzs forskargrupp. Tack för att ni har stått ut med att jag tillbringat så mycket tid i ert pre-PCR-lab! Ett särskilt tack till Sofia som lärde mig grunderna i realtids-PCR, och till Bahram och Henrik för att ni

43 syntetiserade aminoaceton. Hoppas att ert lab inte blev alltför nerkladdat med SSAO-substrat!

Ulla, Marja-Leena, Birgitta och Marita. Det märks när ni är på semester. Tack för att ni håller verksamheten flytande och för att ni alltid är lika hjälpsamma och glada!

Cellbiologen. Tack så mycket för allt ni kunde berätta om diabetes. Ett särskilt tack till Astrid Nordin. Vi hade helt enkelt inte kunnat genomföra somliga av våra försök om vi inte hade fått hjälp av dig.

Mina kära vänner och studiekamrater. Den ”biologiska familjen”, Lena och Jenny. My ”Edinburgh-family”, Cynthia, och Martin. You have all made my studies a pure pleasure! The best friends I could possibly have!

Mest av allt skulle jag vilja tacka min familj. Mamma, Pappa, Jens, Mormor, Ewa, Louise, Felix och Linnéa. Min älskade man Niklas, som i särklass mest idogt korrekturläst min avhandling, och våra härliga barn Alma och Klas. Min värld kretsar kring er! Tack för allt! För att ni är de ni är!

44 REFERENCES

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