Gut microbial translocation in coronary artery disease: Emphasis on physical activity and cardiometabolic disturbances

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UNIVERSITY OF OSLO Faculty of Medicine

Gut microbial translocation in coronary artery disease:

Emphasis on physical activity and cardiometabolic disturbances

Thesis for the degree of Philosophiae Doctor (PhD)

Susanne Kristine Aune, MD

Center for Clinical Heart Research, Department of Cardiology,

Oslo University Hospital, Ullevål

Oslo 2022

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© Susanne Kristine Aune, 2023

Series of dissertations submitted to the Faculty of Medicine, University of Oslo

ISBN 978-82-348-0137-2

All rights reserved. No part of this publication may be

reproduced or transmitted, in any form or by any means, without permission.

Print production: Graphics Center, University of Oslo.

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Table of contents

Acknowledgements ... 5

Selected abbreviations ... 7

List of papers ... 9

Summary in Norwegian ... 10

Summary in English ... 13

1. Introduction ... 17

1.1. Coronary heart disease ... 17

1.1.1. Risk factors for coronary heart disease... 17

1.1.2. The atherosclerotic process ... 19

1.2. Gut microbiota and gut leakage ... 21

1.2.1. The intestinal barrier ... 22

1.2.2. Gut leakage and gut-associated inflammation ... 23

1.2.3. Gut leakage and cardiometabolic risk ... 25

1.2.4. Markers of gut leakage ... 27

1.2.4.1. Lipopolysaccharide (LPS) ... 27

1.2.4.2. LPS-binding protein (LBP) ... 29

1.2.4.3. Cluster of differentiation 14 (CD14) ... 29

1.2.4.4. Toll-like receptor 4 (TLR4) ... 29

1.2.4.5. Toll-like receptor 2 (TLR2) ... 31

1.2.4.6. Intestinal fatty-acid binding protein (I-FABP) ... 31

1.2.4.7. Other markers of gut leakage and microbial metabolites ... 32

1.3. Physical activity and exercise ... 33

1.3.1. Exercise physiology and exercise testing ... 33

1.3.2. Physical activity and cardiometabolic risk ... 35

1.3.3. Physical activity and gut health ... 36

1.4. Gut leakage, physical activity and cardiometabolic health ... 37

2. Aim and hypothesis of the thesis ... 38

2.1. Overall aims ... 38

2.2. Specific aims ... 38

2.3. Hypotheses ... 39

3. Materials and methods ... 40

3.1. Study subjects and design ... 40

3.2. Exercise testing ... 41

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3.3. Exercise intervention ... 42

3.4. Coronary angiography ... 43

3.5. Laboratory analyses ... 44

3.5.1. ELISA methods ... 44

3.5.2. LAL assay ... 45

3.5.3. Gene expression analyses ... 45

3.5.4. Diet registration ... 45

3.6. Statistical analyses ... 46

4. Summary of results ... 47

4.1. Paper I ... 47

4.2. Paper II ... 48

4.3. Paper III ... 50

4.4. Paper IV ... 52

5. Ethical considerations ... 53

6. Discussion ... 55

6.1. Methodological considerations ... 55

6.1.1. Study populations and design ... 55

6.1.2. Measures of circulating gut leakage markers ... 57

6.1.3. Exercise testing and intervention ... 59

6.1.4. Diagnosis of CAD ... 60

6.1.5. Diet and dietary registration ... 61

6.1.6. Other factors possibly influencing the gut leakage markers ... 62

6.2. General discussion ... 64

6.2.1. Gut leakage and physical fitness ... 64

6.2.2. Effect of exercise on gut leakage in CVD ... 65

6.2.2.1. Long term exercise training intervention ... 65

6.2.2.2. A single bout of strenuous exercise; short and long duration ... 67

6.2.2.3. Gut leakage and exercise as related to cardiac biomarkers and CAD... 69

6.2.2.4. Associations with diet and n-3 PUFAs ... 70

6.2.3. Gut leakage in association to cardiometabolic disease states and risk factors ... 71

7. Conclusions ... 75

8. Future perspectives ... 77

9. References ... 78 Papers I-IV

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Acknowledgements

I was first introduced to the field of scientific research when I as a medical student knocked on Professor Ingebjørg Seljeflot´s door to ask if she had any ongoing studies regarding exercise training in the cardiovascular field. She welcomed me enthusiastically and with open arms, and introduced me to the, at that time, ongoing EXCADI trial. The Center for Clinical Heart Research (CCHR) proved to be an exceptionally well organised, functioning, productive and ambitious group of outstanding researchers, and there was therefore no doubt in my heart when I after graduation had the opportunity to come back as a PhD-candidate in 2019.

Ingebjørg then introduced me to a young, aspiring cardiologist and researcher; my main supervisor Ragnhild Helseth, to whom I owe the deepest gratitude. I am in awe of her work capacity, her contagious enthusiasm, her optimism and seemingly never ending knowledge, and I am beyond grateful for her help and support over the last years. The same can be said about Ingebjørg, who has been an invaluable co-supervisor, sharing willingly and enthusiastically from her pool of immense experience. These extraordinary women share many of the same qualities, and I am proud and humble to be a part of their professional sphere. I would also like to thank my co-supervisors Marius Trøseid and Svein Solheim for their help, support and vital contributions, and for their unparalleled expertise in the fields of gut microbial translocation and cardiovascular diseases, respectively.

Furthermore, I would like to thank Professor emeritus Harald Arnesen for his outstanding passion for research. His wisdom is truly inspiring. I am also grateful to Harald for bringing so much humour and joy into everyday work life, and for energetically and vibrantly discussing last weekend´s sporting events with me during lunch, being that of professional athletes or those of our own.

I am also grateful for Jeanette K. Steen and Sissel Åkra, who taught me all I know about laboratory work, and who, together with Vibeke Bratseth, has contributed greatly with sampling and analysing, and with interpretation and helping me understand our results. I whole heartedly thank Charlotte Holst Hansen and Trine Baur Opstad, key pieces of the CCHR, for continuous support, laughs,

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positivity and for creating such a warm and fruitful work environment. You make any day brighter.

And a special thanks to Hilde Fosse, for numerous coffee breaks, conversations, hugs and vitally important minutes in the sunshine outside of Ullevål.

I would like to thank my fellow PhD-students; Miriam, Deji, Are, Hani, Ellen, Aasmund, Vibeke, Jostein, Andraz and Kristine, for interesting discussions, essential support and feedback, coffee breaks and for being a social arena during a couple of somewhat difficult years in pandemic solitude.

I would also like to express my gratitude to Rune Byrkjeland of the EXCADI trial, Arnljot Flaa, Joanna Cwikiel and Charlotte Holst Hansen of the CADENCE study, Kristian Laake and Are Kalstad for my part of the OMEMI trial, and also Peder Myhre and Sjur Tveit, essential for the total OMEMI trial. I am deeply grateful for being able to take part in your studies. I also want to express my gratitude to all other people involved in these trials, and to Sigrun Halvorsen for facilitating clinical research within the Department of Cardiology at Oslo University Hospital Ullevål. Scientific research is truly a team effort, and I am in debt to a lot of people. A special thanks to Jonny Hisdal, and his excellent PhD- students Christoffer Nyborg and Martin Bonnevie-Svendsen, for inviting us to take part in the NORSEMAN trial, an opportunity we value deeply and are very proud of.

I am also very grateful for the financial support and funding from the Stein Erik Hagens foundation for Clinical Heart Research, Oslo, Norway, and the invaluable financial aid given by “Ada og Hagbart Waages Humanitære og Veldedige stiftelse”.

Lastly, I want to thank my dear Fredrik, for being my constant and reliable source of support,

humour, love and joy in everyday life. I cannot wait to start this new chapter with our beloved Aurora soon entering our lives. A special thanks to my two sisters, Benedikte and Henriette, for being my inspiration in everything I do. I also want to thank my parents Kristin and Asbjørn, my mormor Gunn, my mum´s husband Per-Aage, my in-laws Gølin and Anstein, and the rest of my family and friends for all-important love and support along the way – you bring meaning to my life.

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Selected abbreviations

ACS Acute coronary syndrome

ASCVD Atherosclerotic cardiovascular disease AT Ventilatory anaerobic threshold

BMI Body mass index

CAD Coronary artery disease CD14 Cluster of differentiation 14 CCS Chronic coronary syndrome CHD Coronary heart disease

CPET Cardiopulmonary exercise testing CRF Cardiorespiratory fitness

cTnT Cardiac troponin T CVD Cardiovascular disease

ELISA Enzyme-Linked Immunosorbent Assay EST Exercise stress test

HR Heart rate

I-FABP Intestinal fatty-acid binding protein LBP LPS-binding protein

LDL Low-density lipoprotein LPS Lipopolysaccharide MetS Metabolic syndrome

METs Metabolic equivalent of task

NSTEMI Non-ST-elevation myocardial infarction NT-proBNP N-terminal pro-B-type natriuretic peptide PAMPs Pathogen-associated molecular patterns RER Respiratory exchange ratio

RPE Rated perceived exertion sCD14 Soluble CD14

STEMI ST-elevation myocardial infarction TLR2 Toll-like receptor 2

TLR4 Toll-like receptor 4 T2DM Type 2 diabetes mellitus VO2max Maximal oxygen uptake VO2peak Peak recorded oxygen uptake

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List of papers

I. Aune SK, Byrkjeland R, Solheim S, Arnesen H, Trøseid M, Awoyemi A, Seljeflot I, Helseth R: Gut related inflammation and cardiorespiratory fitness in patients with CAD and type 2 diabetes: a sub-study of a randomized controlled trial on exercise training.

Diabetol Metab Syndr 2021; 13:36

II. Aune SK, Cwikiel J, Flaa A, Arnesen H, Solheim S, Awoyemi A, Trøseid M, Seljeflot I, Helseth R: Gut Leakage Markers in Response to Strenuous Exercise in Patients with Suspected Coronary Artery Disease. Cells 2021, 10, 2193.

III. Aune SK, Bonnevie-Svendsen M, Nyborg C, Trøseid M, Seljeflot I, Hisdal J, Helseth R: Gut leakage and cardiac biomarkers after prolonged strenuous exercise. Med Sci Sports Exerc. 2022; doi: 10.1249/MSS.0000000000002948

IV. Aune SK, Helseth R, Kalstad A, Laake K, Åkra S, Arnesen H, Solheim S, Seljeflot I:

Associations between LPS-related gut leakage markers, corresponding gene expression in adipose tissue, cardiometabolic disturbances and dietary habits in patients with a recent MI. In manuscript.

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Summary in Norwegian

Da vi startet arbeidet med dette PhD-prosjektet høsten 2019, ønsket vi å studere livsstilsfaktorer, med særlig vekt på trening og overvekt, og deres relasjon til tarmlekkasjemarkører i populasjoner med og uten hjertesykdom. Det var tidligere vist at personer med koronarsykdom og relaterte metabolske risikofaktorer som metabolsk syndrom, diabetes type 2 og overvekt ofte hadde en ubalanse i tarmfloraen. Denne ubalansen førte til økt lekkasje av bakterieprodukter fra tarmen til sirkulasjonen, noe som var vist å kunne føre til økt generell lavgradig inflammasjon og aktivering av immunforsvaret – en pådriver i sykdomsforløpet for både koronarsykdom og de relaterte metabolske forstyrrelsene. Vi ønsket å undersøke dette i populasjoner med koronarsykdom. Det var også

indikasjoner på at livsstilsfaktorer kunne påvirke lekkasjen av bakterieprodukter, særlig hadde man sett at regelmessig trening kunne påvirke tarmfloraen positivt, og derfor også kanskje påvirke graden av lekkasje. Dette hadde ikke blitt studert hos personer med koronarsykdom. Heller ikke den akutte effekten av trening på tarmlekkasjemarkører var kartlagt, verken hos personer med koronarsykdom eller hos friske atleter.

Vi studerte først tarmlekkasjemarkører i en treningsintervensjonsstudie på pasienter med diabetes type 2 og etablert koronarsykdom, der pasientene ble randomisert til et år med kombinert stryke- og utholdenhetstrening eller kontroll (vanlig oppfølging av fastlege). Totalt 137 pasienter ble med i studien, og 114 fullførte intervensjonen tilfredsstillende og fullførte >60% av treningen. Vi undersøkte sammenhengen mellom fysisk form og tarmlekkasjemarkører, og om

tarmlekkasjemarkørene var påvirkbare av regelmessig trening. Vi fant at baselineverdier av markøren sCD14 korrelerte negativt med maksimalt oksygenopptak, noe som indikerer at de som er i bedre fysisk form har lavere tarmlekkasje. Likevel klarte vi ikke å påvirke noen av tarmlekkasjemarkørene med et års treningsintervensjon, heller ikke i den gruppen som økte sitt maksimale oksygenopptak.

Vi har spekulert i om de rett og slett var «for syke» til å kunne endre nivået av tarmlekkasje med trening, eller om de mange medisinene de brukte kunne maskere små endringer i

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tarmlekkasjemarkører. Vi har også diskutert om vi hadde fått et annet resultat om pasientene hadde klart å trene så mye som de skulle.

I den andre (CADENCE) og den tredje studien (NORSEMAN) studerte vi effekten av en kortvarig eller langvarig treningsøkt på tarmlekkasjemarkører hos henholdsvis pasienter med symptomer på koronarsykdom (Artikkel II) og hos friske atleter (Artikkel III). Vi undersøkte også om nivået av tarmlekkasjemarkører var høyrere hos pasienter som viste seg å ha koronarsykdom i CADENCE, og om nivået korrelerte med utslippet av hjerteskademarkørene TnT og NT-proBNP i NORSEMAN.

I CADENCE ble til sammen 287 pasienter som var henvist til belastningstest grunnet symptomer på koronarsykdom inkludert. I snitt syklet de ni og et halvt minutt til de var utmattet. Vi fant at tarmlekkasjemarkørene sCD14, LBP og LPS steg umiddelbart etter trening, men at markøren på ødelagt tarmbarriere, I-FABP, ikke ble påvirket av denne korte, men intense treningsøkten. Det var ingen forskjell på tarmlekkasjemarkører hos pasienter som hadde koronarsykdom diagnostisert med koronar angiografiundersøkelse sammenlignet med de som ikke hadde koronarsykdom. Vi

konkluderte med at pasienter som har etablert koronarsykdom ikke ser ut til å ha høyere

tarmlekkasje i forbindelse med intens trening enn de som er hjertefriske. I NORSEMAN inkluderte vi 42 friske atleter som fullførte en Ironman fulldistanse triatlon (3,8 km svømming, 180 km sykling og 42 km løping). De brukte i snitt 14 og en halv time på å fullføre løpet. Både hjertemarkørene TnT og NT-proBNP, samt tarmlekkasjemarkørene sCD14 og LBP steg rett etter treningsøkten, før de falt til basale verdier etter en uke. Også markøren på tarmbarriereskade I-FABP steg rett etter trening, noe som indikerer at slik langvarig, intens trening fører til skade på cellene som beskytter tarmveggen.

Det var ingen korrelasjon mellom hjertemarkører og tarmlekkasjemarkører etter trening, og vi konkluderte med at systemisk inflammasjon indusert av tarmlekkasje ikke har noen sammenheng med utslipp av hjertemarkører etter en intens trening hos friske atleter.

I den siste studien (Artikkel IV) undersøkte vi relasjonen mellom tarmlekkasjemarkører i sirkulasjonen og genuttrykket av disse i underhudsfett hos eldre pasienter som hadde gjennomgått et hjerteinfarkt

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2-8 uker tidligere (OMEMI). Studien er basert på et utvalg av 382 pasienter mellom 72 og 80 år. Vi studerte både sirkulerende markører og genuttrykket av disse i relasjon til kardiometabolske

risikofaktorer, samt om nivået av markører var påvirket av pasientenes selvrapporterte kosthold eller inntak av omega-3 tilskudd.

Vi fant at sirkulerende LBP var høyere hos pasienter med overvekt og fedme, samt hos pasienter med metabolsk syndrom, type 2 diabetes, hypertensjon og hos de med lave verdier av HDL-kolesterol og høye verdier av triglyserider. I tillegg var LBP uttrykt i større grad i fettvev hos pasienter med overvekt og fedme, mens TLR2 og CD14 var økt uttrykt i fettvev hos pasienter med metabolsk syndrom og type 2 diabetes. På bakgrunn av dette konkluderte vi med at inflammasjon relatert til tarmlekkasje har en rolle i den systemiske lavgradige inflammasjonstilstanden som er assosiert med kardiometabolske tilstander. Vi fant derimot ingen sammenheng mellom kosthold og nivå av tarmlekkasjemarkører i blod eller i fettvev. Vi fant at LPS i sirkulasjonen var høyere hos de som tok omega-3 tilskudd, noe som var litt overraskende. Likevel er det sett i andre studier at omega-3 faktisk kan øke opptaket av LPS, tatt i betraktning at fettstoffer fasiliterer opptaket av LPS fra tarmen gitt at det ikke er brudd på tarmbarrieren. De som tok omega-3 tilskudd hadde videre lavere uttrykk av inflammasjonsmarkørene TLR2 og CD14 i fettvev, noe som bygger opp under at tilskudd av omega-3 har en antiinflammatorisk effekt.

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Summary in English

When we started this PhD-project in the autumn of 2019, we wanted to study lifestyle factors, with special emphasis on exercise training and overweight, and their association to gut leakage markers in populations with and without coronary artery disease. At the time, it was shown that individuals with coronary artery disease and related cardiometabolic disorders such as the metabolic syndrome, type 2 diabetes and overweight, had an imbalance in their intestinal microbial flora. Such imbalance could lead to leakage of bacterial products from the gut into the circulation, shown to activate the immune system, causing a chronic low-grade inflammation – an important driver of disease progression of both coronary artery disease and related metabolic disturbances. We wanted to investigate this in individuals with coronary artery disease. There were also indications that lifestyle factors could influence gut leakage. In particular, it had been shown that exercise training could modulate the intestinal microbiota positively, and could therefore perhaps also influence the degree of leakage.

This had not been investigated in individuals with coronary artery disease. The acute effect of exercise training had not been investigated either, neither in individuals with coronary artery disease nor in healthy athletes.

First, we studied gut leakage markers in patients with combined coronary artery disease and type 2 diabetes randomised to either one year of exercise training intervention or control (conventional follow-up by their GP). A total of 137 patients were included, of which 114 completed >60% of the training. We investigated the association between physical fitness and gut leakage markers, and whether the training intervention could influence the degree of gut leakage markers. We found baseline values of the gut leakage marker sCD14 to correlate inversely with maximal oxygen uptake, which indicates that those in better physical shape has lower gut leakage. One year of training intervention did not, however, influence the gut leakage markers. We have speculated that the patients may have been “too ill” to benefit from exercise training, or whether the medication they were using might have masked small changes in gut leakage markers. We have also discussed that

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the results might have been different if the patients had managed to exercise as much as they were supposed to.

In the second (CADENCE) and the third study (NORSEMAN), we investigated the effect of one single bout of strenuous exercise of short or long duration on gut leakage markers in patients with symptoms of coronary artery disease (Paper II) and in healthy athletes (Paper III). We also

investigated whether gut leakage markers were increased in patients who proved to have coronary artery disease in CADENCE, and if levels correlated with exercise-induced release of cardiac

biomarkers TnT and NT-proBNP in NOSREMAN.

In the CADENCE study 287 patients referred to exercise stress testing due to symptoms of coronary artery disease were included. On average, they exercised for nine and a half minutes until

exhaustion. We found gut leakage markers sCD14, LBP and LPS to be increased after strenuous exercise, but that the marker for intestinal damage, I-FABP, was not influenced by this short,

strenuous exercise. There was no difference in gut leakage markers between patients who proved to have coronary artery disease diagnosed by coronary angiography compared to those who did not.

We concluded that patients with coronary artery disease does not have increased levels of gut leakage markers in relation to strenuous exercise training compared to those who are healthy. In the NORSEMAN study, we included 42 healthy athletes finishing a full distance ironman triathlon (3.8 km swimming, 180 km biking, 42 km running). They had an average finish time of 14 and a half hours.

Both cardiac biomarkers TnT and NT-proBNP, as well as the gut leakage markers sCD14 and LBP, increased after the race, before returning to baseline values after a week. I-FABP also increased after the race, indicating that such strenuous training of long duration may damage the cells lining the intestinal barrier. There was no correlation between the gut leakage markers and the cardiac

biomarkers, and we concluded that systemic inflammation induced by gut leakage has no association to release of cardiac biomarkers after strenuous exercise in healthy athletes.

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In the last study (Paper IV), we investigated the relationship between circulating gut leakage markers and the corresponding genetic expression of these in subcutaneous adipose tissue of elderly patients with a recent myocardial infarction (OMEMI). The study was based on 382 patients aged 72-80 years.

We studied both circulating levels and the genetic expression in relation to cardiometabolic

disturbances, and whether the levels were affected by the patients´ reported diet and intake of n-3 PUFA supplement.

We found circulating LBP to be higher in overweight and obese patients, in patients with metabolic syndrome, type 2 diabetes and hypertension, and in those with low HDL-cholesterol and high triglycerides. Additionally, LBP was expressed to a higher degree in adipose tissue of overweight and obese, while expression of TLR2 and CD14 were increased in patients with metabolic syndrome and type 2 diabetes. We concluded that inflammation related to gut leakage plays a role in the systemic low-grade inflammatory state associated with cardiometabolic disturbances. On the other hand, we found no association between diet and levels of circulating or genetic expression of gut leakage markers. LPS was higher in patients taking n-3 PUFA supplements, which was somewhat surprising.

However, it has been shown in other studies that n-3 PUFAs actually can increase the translocation of LPS and that fatty acids can facilitate the uptake. Nonetheless, n-3 PUFA supplement was associated with lower genetic expression of TLR2 and CD14 in the adipose tissue, which supports a net anti- inflammatory effect.

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1. Introduction

1.1. Coronary heart disease

Cardiovascular disease (CVD), including coronary artery disease (CAD), cerebrovascular disease, peripheral artery disease and heart failure, is with 17.9 million deaths in 2019 the leading cause of death globally (1), and along with cancer the leading cause of morbidity and mortality in Norway in 2018 (2). Although a decrease in the incidence of CVD has been observed in Norway the last decades, an increasing number of patients are living with established CVD due to increased survival rates.

CAD is the most common type of CVD and is mainly due to the development of atherosclerosis in the coronary arteries, eventually resulting in ischemic damage to the myocardium and hence

development of coronary heart disease (CHD). CHD include chronic coronary syndrome (CCS) and acute coronary syndromes (ACS) such as ST-elevation myocardial infarctions (STEMIs) and non-ST- elevation myocardial infarctions (NSTEMIs). Although essentially different, the terms CAD and CHD are often used interchangeably. The development of atherosclerosis is recognized as a chronic, progressive inflammatory process of the arterial vessel wall (3), described in section 1.1.2.

1.1.1. Risk factors for coronary heart disease

Current guidelines for preventing or limiting progression of CHD concentrate around the established risk factors for atherosclerotic CVD (4). Risk factors are either non-modifiable, such as age, sex and ethnicity, or modifiable. The main modifiable risk factors include smoking, hypercholesterolemia, hypertension, type 2 diabetes mellitus (T2DM) and obesity, and are estimated to be responsible for over half of cardiovascular mortality (5). There is consensus that a large proportion of CHD can be prevented by addressing these behavioural risk factors (4). However, when traditional risk factors are accounted for, there is still some residual risk (6). It is therefore of great importance to identify unknown risk factors, and investigate their modifiability.

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Sedentary behaviour is considered a risk factor for CHD, as well as for developing obesity, insulin resistance and T2DM (7). On the other hand, physical activity can attenuate the risk, and is recommended (7). Exercise and physical activity will be discussed thoroughly in section 1.3.

A healthy diet is another cornerstone in CVD prevention. It reduces the risk for CVD through influencing other risk factors such as lipids, body weight, T2DM and blood pressure. In short, it is recommended to replace saturated with unsaturated fats, reduce the intake of salt, alcohol and free sugars, chose a more plant based diet and eat fish at least once a week (4). Dietary supplements of essential marine n-3 polyunsaturated fatty acids (n-3 PUFAs), or fish oil, for CVD prevention is debated (4). Some large meta-analyses have shown a slight reduction in CV outcomes and mortality, while others have failed to show any effect (8-10). To date, n-3 PUFAs are recommended for high-risk patients who despite statin treatment and lifestyle measures have high triglyceride levels (>1.5 mmol/L) (4, 11).

T2DM has been shown to be associated to CVD and development of atherosclerosis (12, 13). T2DM is defined as individuals who have relative insulin deficiency and peripheral insulin resistance (14), and is diagnosed by having either i) fasting blood glucose ≥ 7.0 mmol/L, ii) a plasma glucose ≥ 11.1 mmol/L in a patient with symptoms or 2 hours after an oral glucose tolerance test or iii) HbA1c ≥ 48 mmol/mol (14). Dyslipidaemia, insulin resistance, T2DM and central adiposity can be classified as intricately interrelated disturbances in metabolic function, and are shared risk factors for

atherosclerotic CHD (15). There has been several attempts at combining these metabolic

dysfunctions defining the metabolic syndrome (MetS) (16), but to date no uniform definition exists (17, 18). Although there are small differences in the details, the definitions agree upon the four major components of MetS: hypertension, dyslipidaemia, glucose intolerance and central adiposity.

Cardiometabolic diseases denotes a combination of these metabolic dysfunctions and the related CVDs, and thus cardiometabolic risk factors are the shared risk factors for developing MetS and CVD.

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Common for the cardiometabolic dysfunctions is a state of chronic low-grade inflammation (19).

Adipose tissue, muscle and liver are sites of inflammation in these individuals, and infiltration of macrophages into these tissues is seen in both animals and humans with obesity, insulin resistance and DMT2 (15). The events initiating the inflammatory processes central to the atherosclerotic process and the metabolic disturbances of adiposity and insulin resistance still remain unclear.

However, a growing body of literature points to an unhealthy gut microbiota accompanied by leakage of gut bacterial inflammatory compounds such as lipopolysaccharide (LPS) into the circulation (gut leakage) as a possible link (20). This is elaborated on in section 1.2.3.

Although the link is still associative and the causality remains elusive, gut leakage seem to play a role in several aspects of the development of cardiometabolic disease (21, 22). The role of gut leakage in CAD and the impact of lifestyle factors, including the role of physical activity, have been investigated and discussed in detail in this thesis.

1.1.2. The atherosclerotic process

Although extensively studied, the atherosclerotic process is not yet fully understood. It is, however, consensus about its multifactorial and complex pathogenesis, which is an intricate interaction between dyslipidaemia, endothelial dysfunction, inflammation, immune cell recruitment and activation, and smooth muscle cell proliferation among others (23).

Atherosclerosis develops in the medium to large sized elastic arteries, and is thought to be initiated by a dysfunction of the endothelium lining the artery wall, making the sub-endothelial space exposed to blood borne atherogenic compounds such as low-density lipoprotein (LDL) cholesterol.

Subsequently to extravasation, LDL cholesterols are modified and oxidized and become pro- atherogenic, pro-inflammatory, cytotoxic and initiate chemotactic processes (23). Recruited monocyte-derived macrophages internalise the oxidised lipoproteins through scavenger receptors, eventually forming foam cells (lipid-loaded macrophages), hallmarks of the atherosclerotic process.

The activated immune system modulates disease progression and recruit T-cells, further destabilising

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the endothelium and initiating a fibroproliferative response driven by smooth muscle cells invaded into of the intima (23). The growing foam cells may die from either apoptosis or necrosis, leaving behind a soft, destabilised lipid core within the plaque. Also smooth muscle cells and endothelial cells on site may die during the progression of atherosclerosis, leaving a fragile fibrous cap over the lipid rich core, contributing to plaque instability, eventually rupture, and increased local thrombogenicity (Figure 1).

Figure 1; The atherosclerotic plaque pathogenesis. © 2019 Hailin Xu et al. (24).

Although the progression of the atherosclerotic lesion is mainly asymptomatic, the plaque may eventually reach a size where it causes symptomatic narrowing of the vessel wall and reduced blood supply to the downstream tissue. If happening in the coronary arteries, such a scenario may manifest clinically as CCS, or “stable angina pectoris”. On the other hand, a destabilised plaque may rupture abruptly or an erosion may occur at the surface of the plaque, exposing the thrombogenic core of the plaque causing a sudden obstruction. Such an acute reduction in blood flow causes acute ischemic damage to the tissue, and manifests clinically as an ACS. An acute ischemic event in which there is evidence of myocardial injury along with typical symptoms, electrocardiographic changes or imaging

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showing new loss of viable myocardium is called an acute myocardial infarction (AMI) (25). AMIs are further categorized into five subtypes, of which atherothrombotic AMIs associated with CAD are defined as type I (25).

As previously mentioned, inflammation is recognised to play a crucial role throughout the progression of the atherosclerotic plaque (26). It is not known what initiates the inflammatory process, although numerous possible stimuli have been identified, most importantly oxidised LDL.

Another suggested inflammatory stimulus is the systemic exposure to remote bacterial products (27).

The reactive enhancement of the local inflammatory response in the plaque has been baptised an

“echo” of the systemic inflammation (28). Indeed, low levels of endotoxins such as

lipopolysaccharide (LPS) has been linked to higher incidence of CVD (29, 30), and experimental endotoxemia has shown that the pro-inflammatory cytokine interleukin (IL)-1 could be induced in arteries affected by atherosclerosis (31). The gut microbiota harbours the by far largest amount of bacterial endotoxins in the human body, and has gained increasing attention as a source of endotoxemia related inflammation in recent years (vide infra) (32).

It is obvious however, that no single inflammatory stimuli is responsible for the inflammation associated with the atherosclerotic plaque and CVD, but that each represents one dimension of this extraordinarily complex pathological process (27). In this thesis, we have focused on the role of gut related inflammatory markers and LPS in CHD.

1.2. Gut microbiota and gut leakage

The human gut contains several trillion microbial cells, and includes bacteria, which are the most abundant, as well as viruses, fungi, archaea and eukaryotes; collectively referred to as the gut or intestinal microbiota (33). An updated estimate shows that the number of bacterial cells residing in the human body is actually close to equal to the number of our own human cells (34). In recent years, we have realized the pivotal role these organisms play in our development and in maintaining health, including development and maturation of the host immune system, influencing host cell

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proliferation and vascularization, providing a source of biosynthesis of energy and vitamins, as well as regulating endocrine functions and neurologic signalling (33). The healthy microbiota also provides protection against pathogens (35).

What denotes a healthy microbiota is yet unclear, however some features such as great bacterial diversity, individual stability and a core functional capacity seem to be common for the well-

functioning gut microbial community (33). The unhealthy gut, on the other hand, is characterized by less diversity and a reduction in functional capacity, and this state of imbalance is commonly referred to as gut dysbiosis (36). The emerging interest in gut dysbiosis has revealed its potential role in the development of a number of chronic diseases, including cancer, autoimmune, neurologic and psychiatric disorders (33). Gut dysbiosis has also been shown to associate with chronic low-grade inflammation, obesity and metabolic disorders (20), all risk factors for the development of CHD (vide supra). Additionally, it has been shown to associate independently with cardiovascular adverse

events and to the development of atherosclerosis (37), as mentioned. However, few studies have been able to explore causality, and the relationship between the gut microbiota dysbiosis and CHD states remains associative (38).

These observations has led to a search for the mechanisms linking gut dysbiosis to remote disease manifestations (33). In this thesis we have chosen not to focus on the composition of the gut

microbiota per se, but to study the leakage of LPS into the circulation and associated inflammation as one possible pathway linking gut dysbiosis to cardiometabolic risk factors such as obesity and insulin resistance, physical inactivity and to the presence of CHD.

1.2.1. The intestinal barrier

In addition to regulating the uptake of nutrients and water, the approximately 400 m2 of intestinal surface of the gastrointestinal tract of humans is in constant contact with the gut microbiota. The microbiota contains both commensal bacteria and pathogenic bacteria, and their metabolites and endotoxins. This means that the intestinal barrier needs to be permeable even to small nutrients and

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molecules, but at the same time tightly restricting the direct contact with the microbiota, and able to mount an appropriate immune response to insults. The intestinal barrier functions as both a physical and a functional barrier. In addition to the single cell layer of epithelial cells called enterocytes, it includes intestinal alkaline phosphatase, antibacterial proteins and IgA that detoxifies endotoxins and bacteria in the lumen, a double mucus layer that provides a physical barrier between the luminal contents and the epithelial cells, and tight junctions between the epithelial cells that limit the paracellular mode of transportation into the circulation (Figure 2) (32).

Figure 2; the intestinal gut barrier. © Endocrine Society 2020, (32).

The intestinal gut barrier is crucial in maintaining normal homeostasis of the gut, and dysfunction of this barrier is associated with both local and systemic consequences related to the translocation of bacterial products to the systemic circulation (32).

1.2.2. Gut leakage and gut-associated inflammation

An intestinal barrier dysfunction denotes the breakdown of the local intestinal defence. The

“common ground” hypothesis is that various factors might affect the gut microbiota negatively, such

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as an unhealthy diet, a sedentary lifestyle, etc., which in turn trigger an increase in gut permeability (33). An increase in permeability leads to translocation of bacterial compounds, mainly LPS from the surface of gram negative bacteria, into the circulation (Figure 3) (32). The leakage of LPS and other bacterial compounds across an impaired gut barrier is called gut leakage, and it will induce a cascade of inflammatory signalling (39). In the literature, several terms are used for this phenomenon.

Endotoxemia is a widely used term for the occurrence of LPS in the circulation. Increased intestinal or

gut permeability or in some cases “leaky mucosa” are other terms describing the thought scenario of an impaired gut barrier and LPS-translocation into the circulation. The term gut leakage will be used throughout the thesis.

Figure 3; gut leakage across the intestinal barrier. © Endocrine Society 2020, (32).

Once LPS is in the circulation, it interacts with LPS-binding protein (LBP) and membrane bound or soluble cluster of differentiation 14 (sCD14) as a potent activator of the MD-2/toll-like receptor 4 (TLR4) -complex (40). The TLR4 is a member of the TLR-family, and recognises pathogen-associated

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molecular patterns (PAMPs), especially LPS (41). The activation of the TLR4 on immune cells initiates downstream pro-inflammatory signalling, described in detail under the TLR4-subheading. This inflammatory response has proposedly been named gut-related inflammation. LPS is also able to activate another member of the TLR-family, the TLR2 (vide infra) (42).

There are various ways to assess increased intestinal permeability, including in vivo monitoring of urine concentration of orally administered probe molecules, or in vitro, using mucosal biopsies (43).

These are time consuming and invasive. Additionally, measurements of tight junction breakdown products in urine (e.g. claudin-3) or measurements of intracellular intestinal fatty-acid binding protein (I-FABP) in the circulation can provide an idea of the integrity of the barrier non-invasively (44). Direct measurement of bacterial products in the circulation (LPS) or indirect measurements of LPS-exposure through measuring anti-LPS antibodies or LPS-related inflammatory markers such as sCD14 and LBP, are other methods to assess the consequences of increased intestinal permeability (45). We have chosen to use a combination of some of these methods, described in the “Materials and methods” section.

1.2.3. Gut leakage and cardiometabolic risk

The first studies that linked intestinal microbiota to obesity were performed in mice. They showed that conventionally raised mice had 42% more body fat compared to germ-free (GF) mice, although the GF mice consumed 29% more chow (46). It was also shown that in mice with diet-induced obesity, chronically increased levels of LPS were related to fat deposition, increase in pro-

inflammatory pathways and insulin resistance (47, 48). LPS-infusion in mice led to the same results (47). These observations were the first to indicate an LPS-related pathway in mediating a pro- inflammatory state in obesity and related metabolic abnormalities. Although not as clear as in mice, several studies have since found elevated levels of LPS in association to obesity, insulin resistance, dyslipidaemia and chronic inflammation in humans (49, 50). LPS has been shown to be able to activate and initiate the transition of the M2 anti-inflammatory macrophages residing in lean adipose

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tissue, into a pro-inflammatory phenotype found in obese adipose tissue (51). Several mediators of the LPS response, including TLR4, CD14 and LBP have been shown to be increased in adipose tissue in association with obesity and metabolic disturbances (52-54), however, the literature is limited.

Importantly, elevated circulating levels of LPS have also shown to associate with incidence of CHD independently of traditional risk factors, C-reactive protein (CRP) and even total energy intake (55).

Also in the development of T2DM, gut leakage seem to play a role. The FINRISK97 study found that higher levels of LPS was associated with an increased incident of T2DM (56). In T2DM, LBP and I-FABP levels were shown to be significantly higher than in healthy controls (57). The activation of TLR4, as well as several of the downstream mediators of inflammation, have been found to interfere with insulin signaling pathways, thus providing a plausible explanation for the link between LPS and the development of insulin resistance and T2DM (58). Elevated levels of LBP have been associated with atherosclerosis, CAD and all-cause mortality (59-61), and sCD14 has been associated with all-cause mortality, CHD and other vascular disease independent of other markers of inflammation and traditional risk factors (62).

Modifying the gut barrier and influencing gut leakage

A dysfunctional intestinal barrier may, as described, have detrimental effects on our cardiometabolic health. Thus, identifying lifestyle factors able to modify and improve the barrier function or reduce gut leakage is of great interest. A healthy diet, especially diets high in fibre and healthy fats, has been shown to positively affect the intestinal barrier (63). Higher levels of short chain fatty-acids (SCFAs), particularly butyrate, either by diet or produced locally by butyrate producing bacterial taxa, seem to benefit the gut barrier by serving as a direct energy substrate for the colonic enterocytes (64). Also, n3-PUFAs have been associated with improved intestinal barrier function (65). Finally, higher levels of physical activity and cardiorespiratory fitness has been associated with greater microbial diversity and an increase in SCFA-producing phyla (66). This is discussed more in detail in section 1.3.3. These are all lifestyle factors recommended for cardiometabolic health, regardless of their impact on the

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intestinal barrier integrity. However, whether these lifestyle factors influence cardiometabolic risk partly via their impact on gut health, or if they are beneficial for both, independent of each other, remains to be elucidated.

1.2.4. Markers of gut leakage

1.2.4.1. Lipopolysaccharide (LPS)

LPS is a part of the outermost membrane of gram-negative bacteria, and it is subdivided into three structural domains; lipid A, a core oligosaccharide and an O-antigen (Figure 4). It is the hydrophobic lipid A structure, the innermost subunit of LPS, which anchors the LPS to the membrane and is the most conserved portion of LPS (39). It is also the most pathogenic; the part of LPS which the innate immune system responds to. The endotoxic potential of LPS depends on the number of acyl chains of the lipid A moiety, where a hexa-acylated subunit elicits a stronger immune response than do those with five or four acyl chains (39). Therefore, not all LPS have the same immunostimulatory potential.

If the O-antigen contains full length O-chains, the LPS is considered smooth, whereas absence or reduction of O-chains renders the LPS rough (67).

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Figure 4; Schematic structure of LPS. Copyright © 2013 Maeshima and Fernandez, (68).

It is estimated that the lumen of the gastrointestinal tract contains > 1 g of LPS (69). Hence, an intact and functioning gut barrier is crucial in protecting us from an otherwise lethal exposure to endotoxin (70). However, small amounts of LPS are thought to pass into the circulation. The translocation of LPS is thought to occur either through paracellular leakage due to disruption of the tight junctions between the enterocytes (vide supra) (32), or trough transcellular uptake alongside absorption of fatty-acids from the diet (71). In the case of the latter they will enter the lymphatic system as part of chylomicrons and bypass the initial hepatic detoxification (72). Gut leakage of LPS can also occur with damage to or destruction of the enterocytes.

The LPS concentration in the circulation of healthy individuals is very low, estimated to be

approximately 5.1 pg/ml, but vary greatly with a range from 0.5 to 65 pg/ml in the literature (73). No standardized reference values for endotoxemia exists to date (21).

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LPS is the only direct measurement of gut leakage, whereas others mentioned are indirect markers of leakage. However, LPS has been shown to have a very short half-life. In mice shown to be 2-4 min, and around 80% of the injected LPS was cleared from the circulation within 5 minutes (74).

1.2.4.2. LPS-binding protein (LBP)

LPS-binding protein (LBP) is a 55-65 kDa polypeptide named after its ability to bind to the lipid A part of LPS (75). LBP is synthesized mainly by hepatocytes (76), but also by intestinal epithelial cells and other cell types (77), and released as an acute-phase protein (78). Circulating LBP has been shown to peak in serum shortly after endotoxemia (12h) and remain elevated for up to 72 hours in healthy humans (79). LBP increases with age and weight independently in the general population (80).

1.2.4.3. Cluster of differentiation 14 (CD14)

Cluster of differentiation 14 (CD14) is a horseshoe shaped myeloid differentiation marker that is either glycosylphosphatidylinositol (GPI)-anchored and thus membrane-bound, or soluble (81, 82).

This 50-55 kDa glycoprotein is found primarily on monocytes and macrophages, although low levels also exist on neutrophils, and can be released into the circulation upon LPS stimulation (83). It is an important cofactor for several toll-like receptors (TLRs) and has the unusual ability to bind to a variety of microbial ligands and PAMPs, including a strong affinity for LPS (84). However, soluble CD14 (sCD14) is also, similarly to LBP, produced by hepatocytes in the liver and considered an acute- phase protein not limited to LPS-exposure (85). sCD14 is able to activate mCD14 negative cells, such as endothelial cells and smooth muscle cells (86).

1.2.4.4. Toll-like receptor 4 (TLR4)

TLRs recognize, as mentioned previously, different microbial structural components; PAMPs, upon which they trigger a downstream cascade of intracellular signalling that results in the secretion of inflammatory cytokines (84). The TLR family in humans now consists of 10 TLRs. Most are single- spanning transmembrane proteins with an extracellular domain, shaped like a horse shoe, and with a helix that links the ectodomain to the intracellular conserved region called the Toll/IL-1 receptor (TIR)

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domain (87, 88). TLR4 was identified in 1997, and predominantly recognises PAMPs from gram- negative bacteria, such as LPS. TLR4 activation is dependent upon accessory molecules which can be classified into three groups; regulatory molecules on the cell surface (e.g. MD-2), regulatory

molecules that directly interact with the TLR4 ligand (e.g. sCD14) and regulatory molecules in the endoplasmic reticulum (89). TLR4 activation initiates downstream signalling pathways, resulting in upregulation and secretion of inflammatory cytokines such as IL-1, IL-6 and tumour necrosis factor alpha (TNF-α), as well as chemokines such as monocyte chemoattractant protein-1 (MCP-1) (Figure 5) (87, 90).

Figure 5; assembly of the LPS-LBP-sCD14 complex, binding to TLR4, and downstream signalling of TLR4 activation. Copyright © 2014, Springer Nature, Plociennikowska et.al, (40).

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TLR4 is most abundantly expressed on immune cells from the myeloid lineage such as monocytes and macrophages (91), but is also expressed in adipocytes (92), coronary endothelial cells and smooth muscle cells in atherosclerotic lesions (93, 94), enterocytes of the gut (95), and is also thought to be expressed by skeletal muscle (96, 97).

1.2.4.5. Toll-like receptor 2 (TLR2)

Another TLR known to recognise LPS is TLR2 (42), although it can be activated by a variety of microbial pathogens, and traditionally is thought to recognise gram-positive cell wall components (98). In addition to be present on monocytes and macrophages, TLR2s are also found on human adipocytes, and has been shown to be 10-fold more highly expressed in subcutaneous adipose tissue (SAT) than TLR4 (99).

There is now accumulating evidence that TLR-dependent signalling can contribute to the development of atherosclerosis (100), and that both TLR2 and TLR4 play an important role in activating macrophages and endothelial cells in the atherosclerotic process (101).

1.2.4.6. Intestinal fatty-acid binding protein (I-FABP)

I-FABP is, contrary to the abovementioned gut leakage markers, regarded as a marker for enterocyte damage and thus a marker of impairment of the gut barrier (102). I-FABP is an approximately 15 kDa intracellular cytoplasmic protein, named after the tissue in which it was first found (103). It is

predominantly found in the enterocytes of the duodenum and jejunum, and in low concentrations in the proximal and distal colon (104), and is involved in the intracellular buffering and transport of long chain fatty-acids (105). As it is an intracellular protein, it is normally not detectable in the circulation of healthy individuals (<0.1 µg/L) (104). In general, fatty-acid binding proteins have a relatively short half-life in the circulation, approximately 11 min (106).

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1.2.4.7. Other markers of gut leakage and microbial metabolites

In addition to the markers mentioned above that are studied in the present thesis, there are a number of other gut leakage markers. Some of these markers are mentioned in the discussion section, where possible pathways are elaborated.

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33 1.3. Physical activity and exercise

«All parts of the body, if used in moderation and exercised in labours to which each is accustomed, become thereby healthy and well developed and age slowly; but if they are unused and left idle, they

become liable to disease, defective in growth and age quickly”.

Hippocrates, 460 – 375 BD

Physical activity has been regarded as health promoting since the birth of modern medicine. Physical inactivity and a sedentary lifestyle is a major health concern globally (107), and also highly relevant in Norway where about 70% of the adult population is categorized as inactive (108). The literature clearly states beneficial effects of physical activity on numerous aspects of health, including risk for CHD (109).

1.3.1. Exercise physiology and exercise testing

Physical activity and exercise training denote two different entities. Physical activity is defined as any voluntary bodily movement that increase the energy expenditure above the basal metabolic rate, while exercise training is considered a subcategory of physical activity, and includes planned activity that is repeated on a regular basis with the purpose of increasing or maintaining health and/or fitness (110). The intensity of physical activity can be estimated from the work load achieved, often referred to as metabolic equivalents of task (METs). 1 MET is defined as the resting energy

expenditure, in other words the amount of oxygen consumed by the body at complete rest, and corresponds to approximately 3.5 ml O2/kg/min (111). A 3 MET activity therefore requires the oxygen of three times the resting metabolic rate; that is 10.5 ml O2/kg/min.

Physical exercise demands an intricate interaction between ventilation, the cardio-pulmonary vascular system and peripheral and pulmonary gas exchange in order to meet the demands of working skeletal muscle (112). Cardiopulmonary exercise testing (CPET) is therefore an informative and well suited objective assessment of not only exercise capacity and cardiorespiratory fitness (CRF), but also of diagnostic information in patients with cardiovascular or pulmonary disease (113).

The body’s oxygen uptake (VO2) is the body’s total oxygen consumption, often reported as total VO2

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in litres per minute (L/min) or relative VO2 (mL/kg/min), and is measured by breath-by-breath gas analysis. Equivalently, VCO2 describes the carbon dioxide output. The most well-known parameter attained during CPET is VO2max, the upper limit of O2 utilization by an individual, reflecting maximal CRF (114). However, not all individuals are able to reach the plateau of VO2 required as a criteria for VO2max, despite increasing workload, and thus the highest VO2 attained during testing is defined as VO2peak (115). In healthy individuals, VO2peak and VO2max will often be the same. Another valuable measure that can be obtained through CPET is the ventilatory anaerobic threshold (AT). AT identifies the threshold where oxygen delivery is unable to meet the increasing metabolic demands, and hence the onset of anaerobic pathways for ATP-production, and is defined as the highest VO2 without a sustained increase in lactate. It is found by identifying the point at which the VCO2 increases more than the VO2 (116).

Absolute intensity

Intensity METs % HR max RPE Borg scale Talk test

Light 1.1-2.9 57-63 10-11

Moderate 3-5.9 64-76 12-13 Breathing is

faster, but compatible with speaking full sentences.

Vigorous ≥ 6 77-96 14-17 Breathing is hard,

incompatible with having a

conversation.

Maximal >17-20 You can no longer

talk, breathing very heavily.

Table 1: Definition of light, moderate and vigorous exercise, modified from the 2021 ESC Guidelines on CVD prevention (4).

To interpret the CPET correctly, it is useful to determine whether an individual has performed at their maximal effort or not. In the present work we have used Borg’s scale of rated perceived exertion (RPE) as a measure of the individuals subjective experience of effort (117). The scale ranges from 6 to 20, where 6 is “no exertion at all”, and 20 is “maximal exertion” (Table 1). Peak heart rate (HR) of the subjects age-predicted maximal HR can also be used. In addition, the respiratory exchange ratio

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(RER), defined as the ratio of VCO2/VO2, is an objective measure of effort, and a peak RER of ≥1.1 is acknowledged as an indicator of good effort (112).

1.3.2. Physical activity and cardiometabolic risk

Early in the 1950s, Morris et.al were the first to demonstrate the reduced risk for CAD associated with physical activity, by comparing the conductors to the drivers of the famous double-decker buses in London (118). Since then, a compelling amount of literature has demonstrated the numerous positive cardiovascular benefits of physical activity (119). Physical activity and regular exercise training reduces the risk of all-cause mortality in CVD populations with approximately 30% (120).

Physical activity is therefore comparable to the most widely used cardio-protective drugs such as statins (121). There is compelling evidence for a dose-response relationship, with the least fit having the most to gain from increasing physical activity (122, 123). It has also been shown that the effect of low CRF is comparable to or greater than that of traditional risk factors such as T2DM or smoking for CVD (124), and that every 1-MET increase in estimated CRF may reduce the risk for major

cardiovascular events with a median of 16 % (125). Additionally, physical activity has a positive effect on several risk factors for atherosclerosis (4). It is now recommended for all adults to achieve 150- 300 min of moderate intensity or 75-150 min of vigorous intensity exercise a week, in addition to resistance exercise twice a week and reducing sedentary time throughout the day (4). The combination of aerobic and resistance exercise has also been shown to be the most effective in improving metabolic function in patients with T2DM (126).

Although rare when considering the absolute numbers, acute, strenuous exercise, is paradoxically associated with an increased risk for ACS and sudden cardiac death (SCD), especially in individuals unaccustomed to physical activity or with established CVD (127-129). While SCD in young athletes often is caused by structural cardiac disorders, > 80% of all SCD is due to CHD in those ≥ 35 years of age (119). The triggering mechanisms are not well defined (130). In healthy athletes, cardiac biomarkers troponin T (cTnT), troponin I (cTnI) and N-terminal pro B-type natriuretic peptide (NT-

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pro-BNP) are frequently elevated after strenuous exercise (131). cTnT is a part of the troponin complex in the contractile apparatus of the cardiomyocytes, and is the preferred biomarker for the diagnosis of myocardial infarction in the clinical setting and primarily regarded as a biomarker for cardiomyocyte necrosis (25). However, moderate amounts of cTnT can leak from the cytoplasm of a stressed cardiomyocyte into the circulation due to membrane damage, and thus elevated levels of cTnT is not necessarily equivalent to cardiomyocyte necrosis (132). NT-proBNP is a bi product of the cardiac prohormone BNP that is released due to cardiomyocyte strain and elevated levels thus reflect reduced cardiac function (133). Whether this reflects physiological responses to endurance exercise or represents subclinical pathological processes in healthy subjects is debated (134, 135).

Despite this, there are no doubts that the benefits of physical exercise outweigh the potential deleterious effects, and physical exercise should in general be addressed and individually prescribed in all CVD patients, both in primary and secondary prevention, with few exceptions (4, 110).

1.3.3. Physical activity and gut health

Some studies suggest that the level of physical activity and fitness is positively associated with gut microbial diversity, and thus a healthy gut (vide supra) (136, 137). In animal studies, exercise seem to alter and restore a normal microbiota (138, 139). In 2014, Clarke et.al were the first to show that physical exercise was positively correlated with increased gut microbial diversity in humans (140). It has, however, proven difficult to fully detangle the effect of exercise training from the effect of the associated diet, and the results therefore vary in healthy individuals and athletes (66).

The mechanisms behind the apparent beneficial effect of exercise on gut microbiota and barrier integrity are complex and not fully understood. As mentioned briefly in section 1.3., the relative increase in bacterial taxa producing SCFAs, which serve as an important energy substrate for the gut epithelium, is one proposed theory (141). Decreased transit time, leading to reduced contact time between the pathogens and the intestinal mucus layer may also positively influence the intestinal function (142). Another theory is the improved local and systemic immune response after repeatedly

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being exposed to small amounts of bacterial compounds (143). Nonetheless, current evidence suggests regular endurance exercise to have a beneficial impact on the gut microbial composition and function (144).

As opposed to with regular exercise, it has been shown that acute, strenuous exercise causes elevated levels of LPS in runners after a race (145, 146). A review revealed that exercise duration > 2 hours seems to be the threshold at which gastrointestinal barrier perturbations manifest (147).

Mechanisms for this temporary impairment by exercise is proposed to be through reduced

splanchnic blood flow induced by the activated sympathetic nervous system resulting in GI ischemia and epithelial ischemia- and reperfusion injury (148). Indeed, it has been shown that reduced splanchnic blood flow correlated with intestinal injury assessed by measurements of I-FABP,

permeability and splanchnic tonometry (149). Acute exercise might also regulate the tight junctions of the enterocytes, leading to a tight junction dysfunction and increased paracellular permeability (150).

1.4. Gut leakage, physical activity and cardiometabolic health

In line with the association between exercise and a healthy gut microbiota described in the section above, an impaired gut barrier and gut leakage seem to be inversely correlated with an active lifestyle. Baseline levels of LPS have been shown to be higher in sedentary than in trained individuals (151). There are indications that levels of gut leakage markers are modifiable by exercise and lifestyle interventions, at least in healthy subjects and insulin resistant or obese patients (152, 153). Whether the positive effects of physical activity and lifestyle interventions on levels of gut leakage markers and on cardiometabolic health interact, or if it reflects two separate beneficial effects that are independent of each other, remains to be elucidated (63). The knowledge on the impact of both short and long term physical activity on gut leakage markers in patients with CVD, and the role of physical fitness in these patients, is scarce or lacking.

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2. Aim and hypothesis of the thesis

2.1. Overall aims

The overall aim of this thesis was to explore gut leakage markers and their association to lifestyle, with main emphasis on physical activity, in populations with known or suspected CAD, with and without T2DM, and in healthy athletes as related to cardiac injury markers. Additionally, we wanted to investigate whether gut leakage markers and their related genes expressed in adipose tissue were associated with cardiometabolic disturbances in patients with CAD.

2.2. Specific aims

I) To explore the relationship between gut leakage markers and cardiorespiratory fitness in patients with combined CAD and T2DM, and to investigate whether a long-term exercise intervention can impact circulating gut leakage markers in these patients (Paper I).

II) To investigate the relationship between gut leakage markers and cardiometabolic disturbances such as the metabolic syndrome, obesity and overweight, in patients with combined CAD and T2DM (Paper I).

III) To explore gut leakage markers in response to acute strenuous exercise in patients with symptoms of CAD, and their relationship to degree of CAD (Paper II).

IV) To explore levels of gut leakage markers during a strenuous triathlon race in endurance trained athletes (Paper III).

V) To investigate any association between exercise-induced increase in gut leakage and cardiac biomarkers in healthy athletes during a strenuous triathlon race (Paper III).

VI) To explore any relationship between levels of gut-related inflammatory markers expressed in adipose tissue as well as circulatory levels and cardiometabolic disturbances in patients with a recent MI (Paper IV).

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VII) To explore any associations between dietary pattern and gut leakage markers, either circulatory or expressed in adipose tissue (Paper IV).

2.3. Hypotheses We hypothesized that

- patients with greater physical fitness would have lower levels of gut leakage markers - levels of gut leakage markers would be reduced by a long-term exercise intervention in

patients with T2DM and CAD

- acute short-term strenuous exercise would increase gut leakage markers, and that they would increase more in patients with pre-existing CAD

- gut leakage markers would increase transiently after acute, long-term strenuous exercise in healthy athletes, and associate to markers of cardiac injury and reduced cardiac function - levels of both circulating and genetic expression of gut-related inflammatory markers would

be elevated in patients with cardiometabolic disturbances, and relate to each other - lower levels of gut leakage markers would be associated with a healthy diet and to dietary

intake of n-3 PUFAs.

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3. Materials and methods

3.1. Study subjects and design

This thesis is based on sub-studies from four study populations, the EXCADI trial (154), the CADENCE study (155), the NORSEMAN study (156) and the OMEMI trial (157). Each will be presented

separately below.

The EXCADI trial (Paper I)

The EXercise training in patients with Coronary Artery disease and type 2 DIabetes (EXCADI) study population were patients with combined T2DM and angiographically verified CAD (n=137), included in a randomized trial investigating the effect of 12 months combined resistance and endurance training on VO2max and glucose control between August 2010 and March 2012 (154). Patients were randomized 1:1 after an initial CPET, fasting blood sampling and clinical investigation, to the exercise intervention or to a control group with conventional follow-up by their general practitioner. The controls were not discouraged to exercise. The CPET and blood samples were repeated after the intervention period. 123 patients completed the study. Patients with the lowest adherence to the training intervention (< 40% adherence) were excluded (n=9) from the per protocol analyses, thus 114 patients were analysed for the intervention effect.

The CADENCE study (Paper II)

The CADENCE study included patients referred to an outpatient exercise stress test (EST) due to symptoms suggestive of CAD (n=327). Patients ≥18 years of age, both genders, with an intermediate or high pre-test prognostic risk score (Morise score ≥ 9 points) were included between December 2011 and October 2017 (158). The initial aims of the study were to investigate the effect of short- term strenuous exercise on troponin T release for the diagnosis of CAD (155). Blood samples were drawn before and within 5 min after finishing the EST. All patients underwent coronary angiography.

For the purpose of the sub-study included in this thesis, patients were grouped according to the degree of coronary artery stenosis.

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41 The NORSEMAN study (Paper III)

The Norseman Xtreme Triathlon race of 2019 is a full ironman distance triathlon (3800m open water swimming, 180km bicycling and 42.2 km running), with a total climb of 5200 metres, from which volunteer participants (n=44) were recruited by email correspondence prior to the race. The

participants were all healthy athletes. Blood samples were drawn before the race, as soon as possible after the race (n=37), the day after the race (n=36) and, in a sub-set of participants available for follow-up (n=9), one week after the race.

The OMEMI trial (Paper IV)

The OMEMI trial was a prospective, randomized, placebo-controlled, double blinded multicentre trial designed to analyse the effect of a 2 year intervention with 1.8 g n-3 PUFA per day compared to placebo on cardiovascular endpoints in patients aged 72-80 years who had undergone an AMI 2-8 weeks prior to inclusion (159). Clinical examination and recordings, fasting blood samples and dietary recordings were carried out at baseline before the randomization. For the present work, baseline data from a subset of patients, all included consecutively at one site (Oslo University Hospital, Ullevål), in which subcutaneous adipose tissue (SAT) samples were available, was used (n=428).

3.2. Exercise testing

A CPET was performed on a treadmill, using a modified Balke protocol (160) in all patients before randomization, and again within one week after the intervention period (Paper I). Participants warmed up at 4% incline at an initial speed of 2.8, 3.8 or 4.8 km/hr, and after three minutes the inclination was increased every 60 sec by 2% to a maximum of 20%. If the participant was still able to keep going, the speed was increased by 0.5 km/hr until exhaustion or until ended by the physician for safety reasons (161). Ventilation, oxygen and carbon dioxide content was measured continuously breath-by-breath by a Hans Rudolph two-way breathing mask (2700 series; Hans Rudolph Inc, Kansas City, USA) connected to a metabolic cart (Vmax SensorMedics, Yorba Linda, USA). VO2peak was defined as the highest average oxygen uptake measured during consecutive 30 sec, and was also included if

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References

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