The effect of saturated –and unsaturated fat on inflammation, blood pressure and glucose metabolism in normal -and obese weight men and women with elevated LDL-cholesterol: A randomized, clinical trial.

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The effect of saturated –and unsaturated fat on inflammation, blood pressure and glucose metabolism

in normal -and obese weight men and women with elevated LDL-cholesterol: A randomized, clinical trial.

Masterthesis by Silje Fjørtoft

Supervisor: Mette Svendsen

Co-supervisor: Tine Sundfør & Kirsten Bjørklund Holven

Department of Nutrition Faculty of Medicine

University of Oslo

November 2017

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Copyright author Silje Fjørtoft Year 2017

Title: The effect of saturated –and unsaturated fat on inflammation, blood pressure and glucose metabolism in normal -and obese weight men and women with elevated LDL- cholesterol: A randomized, clinical trial.

Author Silje Fjørtoft http://www.duo.uio.no

Print: Reprosentralen, Universitetet i Oslo

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Acknowledgement

I would like to thank my supervisors, Mette Svendsen and Tine Sundfør at the Dept. of Preventive Cardiology, OUS, and Kirsten Bjørklund Holven, Dept. of Nutrition, University of Oslo, for their feedback, wisdom and guidance during this project. To Tine; I am very grateful for you letting me be apart of this project, as well as sharing your knowledge and experience with me and always leaving your door open. Thank you, Mette, for your patience and taking the time to review my work and answer my questions.

To my amazing family; mom, dad, father and mother-in-law, and especially my

significant other: I could never have done this without you. Thanks for supporting and comforting me from beginning to end.

I also want to extend my thanks to everyone at the Dept. of Preventive Cardiology.

Thanks to Ragnhild, Lisa, Lise, Tonje, Eli, Tor-Ole, Sasa, Serena and Terje for being welcoming, helpful as well as supportive.

Lastly, I would like to thank my colleague, Ingrid Imset for useful insights, support and a friendly smile throughout this year, it would not have been the same without you

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Abbreviations

AA Arachidonic Acid ADP Adenine Diphosphate ALA Alpha Linoleic Acid

AMPK Adenine Monophosphate Kinase BMI Bodymass Index

CRF Case Report Form CRP C-Reactive Protein CVD Cardiovascular Disease DAG Diacylglycerol

DASH Dietary approach to stop hypertension DBP Diastolic Blood Pressure

DHA Docosahexaeonic Acid ECs Endothelial Cells

eNOS Endothelial Nitric Oxide Synthase EPA Eicosapentaeonic Acid

EVOO Extra Virigin Olive Oil FFA Free Fatty Acid

GLUT-4 Glucose transporter GWAS Genome Wide

HbA1c Glycated Hemoglobin, type 1c

HDL-C High Density Lipoprotein Cholesterol HOMA Homeostatic Model Assessment

HOMA-IR Homeostatic Model Assessment of Insulin Resistance HS-CRP High Sensitivity C-Reactive Protein

ICAM-1 Intercellular Adhesion Molecule-1 IDF International Diabetes Federation IFNγ Interferon Gamma

IL-1 RA Interleukin1 Receptor Antagonist IL-1β Interleukin-1 Beta

IL-6 Interleukin-6 IL-8 Interleukin-8

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IRS-1/2 Insulin Receptor Substrate -1/2 LA Linoleic Acid

LDL-C Low Density LTs Leukotriens

MCP-1 Monocyte Chemoattractant Protein-1 MHO Metabolically Healthy Obese

MI Myocardial Infarction mmHg millimeters Mercury

MUFA Mono Unsaturated Fatty Acids

MUH-NW Metabolically Unhealthy- Normal Weight MUO Metabolically Unhealthy Obese

NCDs Non Communicable Diaseses NF-κB Nuclear Facotr Kappa Beta

NHANES National Health and Nutrition Examination Survey NHANES National Health and Nutrition Examination Survey NNR2012 Nordic Nutrition Recommendations 2012

NO Nitric Oxide

NODs Nucleotide-binding Oligomerization Domain Proteins ox-LDL Oxidized Low-density Lipoprotein

PGs Prostaglandins

PI3K Phosphatidylinositol 3-kinase PKC Protein Kinase C

PPARα Peroxisome Proliferator-Activated Receptor Alpha PPARγ Peroxisome Proliferator-Activated Receptor Gamma PRRs Pattern Recognition Receptors

PUFA Poly Unsaturated Fatty Acid RCTs Randomized Controlled Trial ROS Reactive Oxygen Species S-CRP Serum C-Reactive Protein SBP Systolic Blood Pressure SFA Saturated Fatty Acids

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SREBP-1 Sterol Regulatory Element Binding Protein 1 SSB Sugar Sweetened Beverages

T2DM Type 2 Diabetes Mellitus

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Abstract

Background: Replacing polyunsaturated fatty acids (PUFA) with saturated fatty acids (SFA) have been proven to have a positive effect on cardio metabolic risk factors.

However, research is limited as to if normal and obese weight individuals respond differently to a dietary pattern rich in either PUFA or SFA.

Objective: To study if the effect of PUFA and SFA on metabolic risk factors for CVD;

systolic (SBP) and diastolic blood pressure (DBP), C- reactive protein (CRP) levels, glucose and glycosylated hemoglobin, type A1c (HbA1c) will differ between normal weight and obese individuals

Subjects and methods: Normal (n=43) and obese weight subjects (n=28), including 49 women and 22 men with LDL-C ≥ 3.0 mmol/l were invited to participate in a 6-week intervention study at Section for Preventive Cardiology at Oslo University Hospital. In the normal weight group BMI ranged from 18.7 kg/m² to 25.0 kg/m², in the obese

weight group BMI was between 30.1 kg/m² and 44.0 kg/m². Participants were randomly assigned to either a PUFA or SFA intervention group. Both groups received advise on a heart-friendly dietary pattern, in addition the PUFA group was instructed to eat a minimum serving of margarine (25g), use PUFA sources for cooking and choose low-fat dairy products, while the SFA group was to consume a minimum portion of butter (24g) and choose full fat dairy products as well as using butter for all cooking purposes. Data was collected at baseline, week 2, week 4 and week 6. Multiple regression analyses were made using the differences from baseline to week 6.

Results: At baseline mean (SD) SBP, DBP, glucose, HbA1c and CRP was 119.5 mmHg (14.2), 78.3 mmHg (3.8), 5.3 mmol/l (0.6), 5.2% (0.3), 1.0 mg/l (0.7) in the normal weight group, 123.7 mmHg (11.5), 82.1 mmHg (8.4), 5.7 mmol/l (0.5), 5.5% (0.5), 2.4 mg/l (1.5) in the obese weight group, respectively. There were no significant differences in change in SBP (p= 0.887), DBP (p=721), glucose (p=0.567), HbA1c (p=0.354) or CRP

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(p=0.049) as was reflected within the normal weight PUFA group by a trend for increased CRP levels (0.3 mg/l, (95% CI 0.0,0.6) (p=0.049). The normal weight PUFA group had a significant decrease in SBP -5.9 mmHg (95% CI-2.4,-9.5) (p<0.05) and glucose -0.2 mmol/l (95% CI -0.4,-0.1) (p=0.02). Intervention compliance was evident in a 9.1 E% and 4.1 E% difference in SFA and PUFA within the normal weight group, and a 10.1 E% and 5.4 E% difference in SFA and PUFA in the obese weight group, respectively.

Conclusion: This study does not support the notion that normal and obese weight subjects differ in response to a SFA or PUFA dietary pattern after a 6-week dietary intervention with regards to SBP, DBP, CRP levels, glucose and HbA1c. A trend was seen in the PUFA dietary pattern for an increased level of CRP in the normal weight group compared to the obese weight group. However, within the normal weight group the PUFA dietary pattern also resulted in reductions in SBP and glucose levels. Further research with a sufficient number of participants is needed to fully uncover the benefits of a PUFA dietary pattern.

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1 Introduction

1.1 Cardiovascular disease

Non-communicable diseases (NCDs) cause more deaths than all other causes combined, and are expected to increase to 52 million by 2030. 82% of the deaths are caused by the major four NCDs; cardiovascular disease (CVD), cancer, chronic respiratory diseases and diabetes, 46,2% of these deaths were due to CVD alone, translating into 17,5 million deaths [1, 2]. The WHO defines CVD as disorders of the heart and blood vessels, and includes coronary heart disease (CHD), cerebrovascular disease, peripheral artery disease, rheumatic heart disease, congenital heart disease and heart failure [3]. Despite the fact that CVD mortality has had a steadily decrease since the 1970s, it still remains the leading cause of death in Norway[4]. Approximately 330 000 Norwegians were registered in 2014 as having a form of CVD, of which roughly 11 700 were fatal [5]. Most cardiovascular diseases can be prevented focusing on behavioral risk factors such as tobacco use, unhealthy diet and obesity, physical inactivity and harmful alcohol consumption using preventive strategies [3]. Non-modifiable risk factors such as age, race and gender we cannot alter. However, those already diagnosed with CVD or classified as high risk can benefit from early detection and appropriate counseling [6].

Risk factors we can address through diet; hypertension, obesity, dyslipidemia, insulin resistance and inflammation, are of particular importance in both primary and

secondary prevention strategies.

1.1.1 Atherosclerotic disease

Atherosclerosis is an inflammatory disease that develops in the walls of the major blood vessels in the cardiovascular system. Although the clinical manifestations of the disease appear from the middle age, changes in the atherosclerotic process are evident as early as in childhood and adolescence. Among the risk factors are obesity, hypertension, dyslipidemia, diabetes, smoking, and physical inactivity, often these function

concurrently and accelerate the disease process [7, 8]. Normally, the endothelial cells (ECs) the innermost layer of cells of the blood vessels resist leukocyte adhesion, however the presence of risk factors can initiate the expression of adhesion molecules

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such as vascular cell adhesion molecule -1(VCAM-1) on the ECs causing leukocytes to attach to the arterial wall [9].

VCAM-1 can be stimulated through accumulation of modified lipoprotein particles in the arterial intima, oxidized lipids through nuclear factor-kappa beta (NF-κB) and

proinflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α).

After penetrating the endothelium, monocytes mature into macrophages and increase their expression of scavenger receptors, which allows for further uptake of modified low density lipoprotein (LDL) particles, such as oxidized LDL (oxLDL). Accumulation of cholesterol rich particles leads to macrophages transforming into foam cells. With time the foam cells become trapped within the intima and eventually die, forming a core of necrotic cells, cholesterol crystals and other extracellular material [10]. The

inflammation and pro thrombotic process is furthered by the production of pro-

inflammatory mediators, reactive oxygen species and tissue factor pro-coagulants [11].

Once the atheroma has formed, smooth muscle cells (SMCs) from the tunica media are recruited into the tunica intima and proliferate in response to growth factors. SMCs residing in the intima produce extracellular matrix molecules such as collagen,

proteoglycans and elastin, forming a fibrous cap that covers the plaque. While SMCs and collagen play a vital part in plaque stability, interferon gamma (IFNγ) and matrix

metalloproteinases inhibits SMC proliferation and differentiation, in addition to collagen fiber degradation [10].

A thin fibrous cap and an inflamed lipid core characterize unstable plaques. Symptoms of atherosclerosis become apparent when the cap no longer can withstand the force from the BP and ruptures, causing blood to come in contact with the lipid core, resulting in thrombus formation and if severe enough possibly a blocked artery [9].

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1.1.2 Obesity

The obesity epidemic has reached epidemic proportions, its prevalence in both developed and developing countries is evident, and affects all socioeconomic classes, genders and ages [12]. According the WHO obesity has more than doubled since 1980. In 2014 more than 39% of adults were overweight, and 13% were obese [13].

In Norway, numbers from Folkehelseinstituttet after year 2000 found that about 20%

of men and 17% of women in the age group 40-45 were obese [14]. Coinciding with the obesity epidemic, life expectancy has also increased, meaning people live longer with diseases such as CVD and diabetes [15]. Not only does this impose a threat to our health, but also the economic burden on our society due to lost productivity and increased costs should be taken into account [2] It is therefore vital that those strategies that have the greatest impact are researched and implemented.

Body mass index (BMI) is a tool frequently used by researchers and healthcare professionals to determine overweight and obesity. It is defined as body weight in kilograms divided by the height in meters squared (kg/m2). Based on these numbers one is allocated into one of five categories (Table 1). BMI is independent of age, race and gender, but does not take into account fat distribution or body composition and is not necessarily a good tool to classify individuals. However, it does serve its purpose at population level.

Table 1. BMI classification according to the WHO

BMI Nutritional status

< 18.5 Underweight

18.5–24.9 Normal weight

25.0–29.9 Pre-obesity

30.0–34.9 Obesity class I

35.0–39.9 Obesity class II

> 40 Obesity class III

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1.1.3 Metabolically Healthy Obese

Obesity is defined as excessive fat accumulation that may impair health [13]. The results are premature mortality, increased morbidity from diseases such as CVD, diabetes and cancer, and reduced quality of life [2]. However, up to 30% of obese individuals are metabolically healthy, meaning they don’t show the obesity-associated complications most frequently observed. This obese phenotype, free of metabolic complications have been termed the metabolically healthy obese (MHO). The pathophysiology of the MHO is as for overweight and obesity a combination of genetics, the environment and behavior.

Just like healthy normal weight individuals they are insulin sensitive, have a low

incidence of hypertension, both satisfactory lipid profiles and fat distribution, and a low level of systemic inflammatory responses compared to most of the metabolically

unhealthy obese who are at an increased risk for developing comorbidities [16,

17]. MHO is usually defined using parameters such as BP, lipid profile, fasting glucose levels, homeostasis model assessment (HOMA), and systemic inflammation using high- sensitivity C-reactive protein levels (CRP) [17].However, no consensus has been reached regarding a definition of the MHO, making it difficult to compare results between studies [18]. It is also worth mentioning that the definitions of MHO are only related to

metabolic –and cardiovascular complications, other obesity-associated difficulties such as orthopedic problems, pulmonary complications, or other physiological conditions are not taken into account [19].

A report from the Framingham offspring study found that obese subjects with a MHO phenotype were not at a substantially increased risk of developing type 2 diabetes mellitus (T2DM) or CVD compared to subjects with the metabolic syndrome regardless of BMI status [20]. However, it has been argued that a 7 year follow-up is not sufficient time to see the long term effects [21]. A 20-year follow-up study looking at the impact of BMI on the metabolic syndrome and the risk of diabetes in middle-aged men, not

surprisingly found obese participants with insulin resistance to have the highest risk.

Nevertheless, overweight and obese men without metabolic syndrome or insulin

resistance also had a significantly increased diabetes risk [21]. Another recent follow-up

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normal weight and the unhealthy obese, and according to long-term studies, MHO could be a transient stage before being defined as metabolically unhealthy obese (MUO).

1.1.4 Metabolically Unhealthy Normal Weight

In contrast to the MHO phenotype, there is a subgroup of normal weight (BMI< 25 kg/m²) patients that also present with increased CVD and metabolic risk; the metabolically unhealthy normal weight (MUH-NW) [23]. This group of patients is usually described as being normal weight with the addition of 2-3 metabolic

abnormalities. However, as for the MHO no standard definition exists, making it difficult to assess the prevalence of this phenotype. In a review of the National Health and

Nutrition Examination Survey (NHANES) from 1999-2004, Wildman et. al. found 23.5%

of normal weight adults to be metabolically abnormal (≥ 2 metabolic abnormalities) [24]. Despite having a normal BMI, they have more abdominal adiposity and a higher prevalence of dyslipidemia compared to their lean healthy counterparts, which leaves the MUH-NW at an increased risk of diabetes mellitus, CVD and mortality [18, 21].

Since treatment and prevention of obesity related diseases takes a great toll on the healthcare system and the economy, it is important to target the group of obese patients that will have the best effect from lifestyle changes or pharmacological interventions.

1.1.5 Metabolic Syndrome

Metabolic syndrome is defined as the clustering of CVD risk factors; hypertension, glucose intolerance, abdominal obesity and dyslipidemia. According to a report from the International Diabetes Association about 20-25% of the world’s population have the metabolic syndrome, making them twice as likely to die from a heart attack or stroke and five times as likely to develop T2DM compared to their healthy counterparts [25].

According to the International Diabetes Federation (IDF) the definition for use in clinical practice for the metabolic syndrome is defined as having central obesity; defined as waist circumference with ethnicity specific values or a BMI>30 kg/m², in addition to at least two of the following factors; triglycerides (TG) ≥ 1.7 mmol/l, reduced high density lipoprotein cholesterol (HDL-C); ≤1.0 mmol/l (men) and ≤1.3 mmol/l (women), systolic BP ≥130 or diastolic BP≥85 mmHg or treatment of previously diagnosed hypertension and fasting plasma glucose ≥5.6 mmol/l.

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1.2 Blood pressure

BP is defined as the force exerted per unit on the walls of the arteries, and is dependent upon the peripheral resistance, the elasticity of the vessels, blood volume and

cardiac output. BP is measured by two numbers, the upper number is the SBP and is the force exerted on the vessels as the heart contracts. The lower number the DBP measures the force as the heart relaxes between contractions. BP is measured in millimeters of mercury (mmHg) [26, 27].

1.2.1 Hypertension

Adult BP is considered normal at SBP of 120 mmHg and a DBP of 80 mmHg. However, there are also cardiovascular benefits to a SBP as low as 105 mmHg and a DBP of 60 mmHg. Normal BP is essential for optimal function of the heart, brain and kidneys as well as for general health. Hypertension is defined as SBP equal to or above 140 mmHg and/ or a DBP equal to or above90 mmHg [27]. For a correct diagnosis of hypertension, BP needs so be measured twice a day for several days, preferably in the morning and evening. Two consecutive measurements, with at least a minute apart provide the basis for the average value, which can either confirm or reject the diagnosis [27]. Ambulatory BP is considered the gold standard for research purposes and is a requirement to confirm the office diagnosis [28]. The measurement process itself can also be stressful and could result in white-coat hypertension, where blood pressure readings are higher than normal when measured in a medical environment. Some studies have found that white-coat hypertension occurs in up to 30% of subjects with elevated office blood pressure [29]. Although not free from the white-coat syndrome, ambulatory blood pressure gives a presentation of the daily blood pressure variations through repeated measurements. However, the chosen day with ambulatory blood pressure

measurements might not be a representative day of the subject’s life, thus causing a potential measurement error.

There are two main groups of hypertension; essential and secondary. About 90% of

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unknown. However, the interaction between genetics and environmental factors play a major role. Secondary hypertension, less commonly known as inessential hypertension, is caused by an identifiable primary cause, including kidney disease, crushing’s

syndrome, tumors, and side effects of certain medications [30, 31].

Table 1. BP classifications

*Values adopted from NICE guidelines [32].

1.2.2 Prevalence

Hypertension is one of the most important independent risk factors for CVD and the most prevalent disease worldwide [33]. According to the WHO, hypertension is responsible for at least 45% of deaths due to heart disease, and 51% of deaths due to stroke. Raised BP is also the leading preventable risk factor for premature death and disability in the world. In 2000 an estimate of 26.4% or 972 million people had

hypertension, 10 years later the numbers had increased to 31 % or 1.39 billion people [34, 35]. Since the turn of the century the prevalence have been increasing in low –and middle-income countries, while high-income countries have had a steady decrease [27, 34]. Based on data from 2007-2010, approximately 78 million American adults age 20 or older have hypertension, about 82% of them are aware of their condition and 75 % are being treated with antihypertensive medication, however only 53% have it under control [36]. Estimates from 2013 show that by 2030, the prevalence of hypertension is projected to increase 7.2 % in the American population [37].

Data from the second healthsurvey in Nord-Trøndelag (HUNT 2), found 44.9% of the population to have hypertension [38]. The prevalence of hypertension in Sweden and Finland during the same time period were 38% and 49%, respectively [39]. Defining hypertension as 140/90, at least 950 000 Norwegians are classified as hypertensive, or about 59% of adults over age 40 are in need of treatment [40].

BP classification* SB (mmHg) DB (mmHg)

Normal <120 <80

Pre-hypertension 120-139 80-89

Stage I Hypertension 140-149 90-99

Stage II Hypertension ≥180 ≥110

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Symptoms

Hypertension is also called the silent killer, as most people with a hypertensive diagnosis have no symptoms. Some individuals might experience symptoms such as headache, shortness of breath, dizziness, chest pain, palpitations of the heart and nose bleeds [27].

Although the presence of these symptoms should be taken into account, they are not sufficient to diagnose hypertension. Chronic hypertension should be taken seriously as it eventually can cause complications such as aneurysms, chronic kidney disease, cognitive changes, eye damage, heart attack, heart failure, peripheral artery disease and stroke [41]

1.2.3 Risk factors influencing BP

Age

With age the structure and function of the heart and vasculature change. Changes in the structural integrity of the vascular wall cause loss of arterial elasticity and reduced compliance and can potentially lead to age-related changes in BP [42, 43]. On average, SBP rise with age, while DBP increases until the age of 50 and declines thereafter [44].

Elevated SBP or DBP is associated with an increase in CVD risk; starting at 115/75 mmHg the risk of CVD doubles for each increment of 20/10 mmHg of BP among middle aged and elderly persons [45].

Genetics, gender and race

As hypertension is a multifactorial and complex disease, several loci have been identifies through linkage studies and geome-wide associstion studies (GWAS). About 40-50% of variability in BP is heritable, however, the associated genetic variation identified so far only explains about 2%. The potential identification of genes regulating BP could help explain the pathophysiology as well as finding new therapeutic methods [46].

Men are largely at a greater risk for developing CVD when compared to age-matched premenopausal women. Data from NHANES III showed men having higher BP than

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Danish men and women, found men to have about 6-10 mmHg higher MAP than women until the age of 70-79 years, thereafter blood pressure values were similar across both genders [49].

Although high BP affects the whole population the prevalence of high BP among African- American men and women are higher compared to their Caucasian counterparts. Stroke and end-stage renal disease risks are two and five times greater for African-Americans men and women, respectively [50]

Obesity

Obesity is a well-known risk factor for hypertension, and the prevalence is

approximately twice as a high compared to nonobese [51]. With obesity comes an increase in blood flow, vasodilation, cardiac output, glomerular filtration rate and hypertension. Renal sodium retention also increases, thereby leading to hypertension.

The factors responsible for these alterations in obesity include enhanced sympathetic tone, activation of the renin-angiotensin system (RAS), insulin resistance, structural changes in the kidneys and the increase of adipokines such as leptin[51, 52]

Insulin resistance

Hypertension is often seen with insulin resistance, and precedes a diabetes diagnosis [53]. Hypertensives have a 5-year risk for developing T2DM that is 2.5 greater than normotensives when controlling for age, sex, and race [54]. A cross sectional multicenter study of 420 essential hypertension patients found a 68% prevalence of abnormal

glucose metabolism [55]. Although evidence of a relationship between an abnormal glucose metabolism and hypertension exists, the exact mechanism behind the

pathophysiology is not fully understood. The association could also be explained by the two factors being symptoms of a metabolic disorder or impaired glucose metabolism could be viewed as a genetic marker of symptoms associated with hypertension[56]. In normal conditions insulin binds to cell surface receptors causing autophosphorylation and activation of insulin receptor substrates (IRS). IRS-1 and IRS-2 are activated through tyrosine phosphorylation, which in turn activates phosphatidylinositol 3-kinase (PI3K).

Activated PI3 then activates protein kinase B leading to the production of nitric oxide (NO) through phosphorylation of NO synthase and relaxation of vascular smooth muscle

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cells. This signaling pathway is impaired in obese and/or insulin resistant persons, causing reduced vasorelaxation and resulting in increased BP [53, 57].

Physical inactivity

The position stand of the American College of Sports Medicine on exercise and

hypertension recommends at least 30 minutes of aerobic endurance training daily [58].

Endurance training lowers BP by reducing the systemic vascular resistance, in which both the sympathetic nervous system and the renin angiotensin system seems to play a part [59]. A Meta analysis looking at the effects of chronic dynamic aerobic endurance training found that training lowered BP in hyertensives by about 7 mmHg systolic and 5 mmHg diastolic. Aerobic training was also found to have a favorable effect on other cardiovascular risk factors [60].

Dietary risk factors

It is a well-known fact that dietary factors play a vital role in the development and progression of hypertension. Both macro and micronutrients have been the focus of several randomized control trials (RCTs).

Sodium

Sodium is needed for several metabolic processes in the body and includes the

maintenance of the extracellular fluid volume, acid base balance, transmission of nerve impulses and nutrient uptake [61, 62]. One g of salt equals about 0.4 g of sodium, and 1 g of sodium equals 2,5 g of salt. A consumption of 1.5 g of salt is set as the lower intake level, also accounting for variations in physical activity and climate. [61].

The relationship between sodium and hypertension has been extensively studied. Cross- sectional population studies have shown a very low incidence of hypertension in

population with very low salt intake (<2 g/d), whereas areas of high salt intake (30- 35g/day) have reported severe hypertension among 30-35% of the population [61]. The Epic Norfolk (the European Prospective Investigation into Cancer in Norfolk) study with 23,104 men and women aged 45 years to 79 years found a significant relation between

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from 10 to 5 g per day would reduce stroke and CVD rates by 23% and 17% respectively [64]. Given the relationship between sodium and BP and what is achievable on a

population level, The Nordic Nutrition Recommendations 2012 (NNR2012) recommends an intake of 2,4 g of sodium/day or about 6 g of salt/ day [61].

Potassium

While potassium is the most abundant cation inside the cell, extracellular potassium is an important regular of BP, acid-base balance and muscle function. Important dietary sources are potatoes, fruits, berries, vegetables and dairy products [61]. The Intersalt study observed an inverse relationship between BP and potassium excretion, while also finding a 30-45 mmol increase in urinary potassium excretion was associated with a 2-3 mmHg lower BP [61, 63, 65]

Fish oil

Fish oils contain the omega-3 fatty acids eicosapentaeonic acid (EPA) and

docosahexaeonic (DHA), known precursors of prostaglandins and thromboxanes. A randomized controlled trial in three European countries of overweight and obese adults found that a 8-week energy restricted diet with three fatty fish meals a week was

associated with reduced BP [66]. Several studies have found effects of omega-3 fatty acid supplementation on BP. In a meta analysis of 31 placebo-controlled trials, there was a reduction of -0.66/-0.35 mmHg per g of ω-3 fatty acid, with the greatest effect on hypertensive subjects [67]. Comparing normotensives and hypertensives, an average intake of about 3 g of ω-3 fatty acids a day, produced reductions by -1.0/-0.5 and - 5.5/3.5 mmHg in the two groups respectively [68]. The BP lowering effect of ω-3 fatty acids may function through increasing the release of nitric oxide (NO), their

incorporation into cellular membranes, the release of adenosine diphosphate (ADP) and the release of prostanoids [69].

Alcohol

A causal relationship between chronic alcohol consumption (>30 g alcohol/day) and hypertension has been established in both clinical and epidemiological studies [70, 71].

When comparing heavy alcohol drinkers to others, the Framingham study reported a 7 mmHg increase in mean arterial pressure (MAP) [72]. Reports indicate that every

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increase of 10 g of alcohol a day is related to an increase of 2 mmHg and 1mmHg in systolic BP and diastolic BP, respectively [73]. In a metanalysis of 16 prospective cross- sectional studies a linear relationship between alcohol consumption and hypertension was observed among the men. The women however, had a J-shaped curve indicating that a moderate intake lowered risk of hypertension while a intake of more than 20g of alcohol a day significantly increased the risk [74].

Dietary fatty acids

Total fat in a dietary pattern is a variation in quantity of saturated fat, ω -3 polyunsaturated fat, ω -6 polyunsaturated fat, and monounsaturated fat. Dietary interventions reducing total fat have a beneficial effect on BP [75], however recent research has focused on the effect of individual types of fat. Epidemiological and intervention studies indicate that a high intake of SFAs and a low intake of PUFAs are associated with a higher BP [76].

In the seven countries study, intake of saturated fat was strongly correlated to the incidence of CVD, total fat however, did not show as strong of a correlation. While Finland had the highest rates of coronary heart disease, Greece was on the opposite end of the scale, noteworthy both countries had about 40% of energy from fat [77]. Similar data was found in the Nurses’ Health study, they also discovered replacing 5 percent of energy from saturated fat with energy from unsaturated fats was associated with a 42%

lower risk of CVD [78]. In a parallel multicenter study, subjects were assigned to one of to isoenergetic dietary patterns; either one rich in monounsaturated fatty acids (MUFA) or a second rich in saturated fatty acids. With the MUFA dietary pattern, SBP and DBP decreased significantly by 2.2 % and 3.8% respectively, no change was seen in the SFA group. Surprisingly, the difference in SBP and DBP by the MUFA dietary pattern

disappeared when the total fat intake was above the median of 37% [79]. Mensink and colleagues looked at the effect of a low-fat, carbohydrate rich dietary pattern compared to a high fat, olive oil-rich dietary pattern on BP in normotensive men and women. Both dietary patterns had the same level of saturated and polyunsaturated fatty acids. There was no effect on BP from the two dietary patterns relative to one another, suggesting

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patterns on BP [80]. However, the subject population was young, normotensive, nonobese, and healthy, thereby making it difficult to alter BP.

LA is the major polyunsaturated fatty acid in the Western dietary pattern. The

INTERMAP study (International Study of Macro-Micronutrients and Blood Pressure) found a non significant inverse relationship between LA intake and SBP and DBP, with a 2-SD higher intake of LA the estimated SBP and DBP differences were -1.42 mmHg and - 0.91 mmHg, respectively [76]. The systemic review (SR) of Schwab and colleagues, including 14 RCTs on the quality and amount of dietary fat on BP, concluded that the evidence for an effect was limited [81].

1.2.4 Nutritional Therapy

A patient with hypertension is more likely to be overweight or obese, have an unhealthy dietary pattern, smoke and/or drink excessively [82, 83]. Although hypertensive

medications are important in treating elevated BP they also come with side effects. Also, those with BP 130/85 do not necessarily qualify for BP lowering medications, but lifestyle modifications have been shown to decrease BP, while also improving the effect of antihypertensive medication [84].

Dietary Approach to Stop Hypertension (DASH)

The DASH diet is a dietary pattern rich in fruits, vegetables, whole grains and low- fat dairy products and includes meat, fish, poultry, nuts and beans, limits sugary foods and beverages and red meats. The DASH trials were a series of controlled trials conducted in the 1990s to look at the benefits of a healthy dietary pattern among men and women.

The DASH multicenter trial found that the dietary pattern lowered SBP by 5.5 mmHg and DBP by 3.0 mmHg in subjects with a BP less than 160/80-95 mmHg [85]. The subjects kept a stable weight, a sodium intake of about 3 g a day and two or less units of alcohol a day. Although the reduction in BP was more prominent in hypertensives, there was a significant reduction in normotensives as well. In subjects with hypertension, the DASH diet produced similar results as those observed in drug monotherapy trials for mild hypertension. Thus leading the researchers to conclude that the DASH diet not only is a preventive measure but may also serve as an alternative to drug therapy in patients with stage I hypertension [85]. As the DASH trial looked at a dietary pattern and not a single

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factor, the conclusion as to why BP decreased significantly compared to a typical American dietary pattern is like the etiology of hypertension itself; multifactorial.

However, as a whole the DASH dietary pattern contains less saturated –and total fat and cholesterol and more potassium, calcium, magnesium, dietary fiber and protein

compared to the typical western dietary pattern.

Mediterranean Diet

It was from Keys’ Seven Countries Study in the 1950s and 60s the Mediterranean dietary pattern, more specifically the unsaturated fats, received recognition for its’ prevention of CVD [86]. The Mediterranean dietary pattern is characterized by a high consumption of fruits, vegetables, whole grain cereals, and fermented dairy products in addition to large amounts of monounsaturated fats predominantly from olives and olive oil. Fish, poultry, nuts and legumes are consumed weekly, while red meats and alcohol (<30g/day) are consumed moderately. Since the Seven Country study several studies have established the Mediterranean diet as beneficial for health status; a more preferable lipid profile, better insulin sensitivity and increased quality of life [87].

The PREDIMED trial is a randomized controlled trial with close to 7500 men and women who had a high risk of developing CVD. The included subjects had no CVD at baseline, but had diabetes type 2 or at least three additional risk factors; smoking, hypertension, elevated LDL-C levels, low HDL-C levels, overweight or obesity, or a family history of premature coronary heart disease. The participants followed one of three energy- unrestricted dietary patterns for about 4 years; a low-fat control diet, a Mediterranean diet with extra virgin olive oil (1 liter per week), or a Mediterranean diet supplemented with 30g of unsalted nuts (a mix of hazelnuts, almonds and walnuts) per day. The Mediterranean dietary pattern with either extra virgin olive oil or nuts gave a relative risk reduction of about 30% in cardiovascular events. Of the cardiovascular end points, only stroke was significantly reduced in both the extra virgin olive oil and nut -

Mediterranean dietary patterns [88]. As there is a close link between BP and stroke, the Mediterranean diet may exert its effect through modifying BP.

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not likely due to the fact that the dietary pattern had the same components as the Mediterranean dietary pattern except from live oil and nuts. Reductions in SBP were greater in two the Mediterranean dietary patterns compared to the control diet, in the extra virgin olive oil group SBP was reduced−1.53 mmHg and 0.69 Hg, respectively [89].

Nordic dietary pattern

The Sysdiet study was a multicenter randomized control study lasting 18-24 weeks comparing an isocaloric healthy diet based on the Nordic nutrition recommendations with an isocaloric control diet formed on the basis of the mean nutrient intake in the Nordic countries. Subjects had a BMI between 27-38 kg/m² in addition to two other criteria for Metabolic Syndrome. The healthy Nordic diet emphasized whole-grain products, berries, fruits and vegetables, rapeseed oil, three fish meals per week, low-fat dairy products and avoidance of sugar-sweetened products. The two diets differed primarily in amount of dietary fiber, salt and the quality of dietary fat [53, 90]. As

opposed to what was observed in the DASH diet study, no statistical difference in BP was observed, however the fat intake was higher and no focus was put on consuming low- salt food items. In a sub-study of the Sysdiet study, 37 subjects with characteristics of the metabolic syndrome lowered 24-h DBP after 12 weeks by 4.4 mmHg and MAP by 4.2 mmHg when following the healthy Nordic diet. Those in the healthy Nordic diet group increased their intake of PUFA, dietary fiber, potassium and protein, while also

decreasing the amount of SFA, sucrose and salt consumed [91].

The NORDIET study, a Swedish randomized control study with 88 participants, looked at the effect of a healthy Nordic diet compared to a control diet with a typical western dietary pattern on cardiovascular risk factor in hypercholesterolemic subjects with BMI

≥20 and ≤31 kg/m². The Nordic diet was based on the Nordic nutrition

recommendations from 2004, while also drawing inspiration from the DASH and- Mediterranean diet, among others. High fiber food items, fruits, berries, vegetables, whole grains, rapeseed oil, nuts, fish and low-fat milk products, and a low salt, added sugar and saturated fat content characterized the Nordic diet. SBP was significantly reduced by 6.6 mmHg in the Nordic diet group, DBP however, did not show the same effect [92].

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1.3 Inflammation

Inflammation occurs as a result of the body’s natural response to an infection or an injury. The inflammatory response includes interactions between cells and the

production of, and responses to a wide variety of chemical mediators. The cardinal signs of inflammation are heat, redness, swelling, pain and loss of function. The acute

inflammatory response is self-regulated through negative feedback mechanisms by secretion of anti-inflammatory cytokines, suppression of pro-inflammatory signaling cascades, loss of receptors for inflammatory mediators, and activation of regulatory cells [93]. A controlled and regulated response is beneficial to maintain homeostasis,

however, when the regulatory processes become unwarranted, tissue damage and disease can occur.

Many of the acute inflammatory characteristics continue as the inflammation becomes chronic. The characteristics of a inflammatory response includes loss of barrier function, a substantial infiltration of inflammatory cells into compartments which normally only contain them in low numbers, overproduction of oxidants, cytokines, chemokines, eicosanoids and matrix metalloproteinases [94]. Chronic inflammation is not only considered a significant contributing factor to several chronic diseases such as CVD, diabetes, Alzheimer and cancer [95], but does in many cases also maintain and advance the disease.

1.3.1 Obesity and inflammation

There is a close link between immunity and metabolism, both under and over nutrition affects the immune function. Subjects with obesity, diabetes, and or CVD has of lately been characterized as having a chronic low-grade inflammation status, and as a consequence there are increased levels of inflammatory markers in their circulation [96]. The concentrations of pro-inflammatory mediators are higher in obese than in normal-weight individuals, for most mediators the variation in concentration is at least 10-fold [96]. Data indicate that the association between BMI and waist circumference and CRP in addition to other inflammatory markers such as interleukin 8 (IL-8) and monocyte chemoattractant protein (MCP-1) is a strong one [97].

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The first molecular link between obesity and low-grade inflammation was discovered over a decade ago, when the pro-inflammatory cytokine TNF-α was identified as

synthesized and released by white adipose tissue in obese rodents. Experiments found, that overproduction of TNF-α in adipose tissue was not only a central feature of obesity but also an important contributing factor to insulin resistance. With time, it became obvious that obesity is characterized by a wide inflammatory response; alongside that of TNF-α several inflammatory mediators show similar response patterns in obesity [98].

Accumulation of adipose tissue can either be centered in the thorax or abdominal area, which is associated with increased risk of diabetes and atherosclerosis, or be located in the lower part in the body, which presents with a lower risk [99]. An increase in the abdominal fat mass is therefore coupled with an increase with several acute-phase proteins such as CRP, pro-and anti-inflammatory cytokines, adhesion molecules and pro- thrombotic molecules. The liver and the lymphoid organs are usually the primary

productions sites for these mediators, but in obesity, and abdominal obesity in

particular, adipose tissue becomes the main production site causing a chronic state of inflammation [96].

Further research lead to the theory that adipose tissue was not only an energy store, but also a vital part of the immune system as a source of pro-inflammatory mediators, which play an important role in chronic inflammation, insulin resistance and atherosclerosis.

With the discovery of leptin, adipose tissue is today recognized as a metabolically active tissue that secretes adipokines as well as pro- and anti-inflammatory proteins [98, 100].

However, adipocytes are not the sole producers of adipokines, macrophages that reside in the non-adipocyte fraction of adipose tissue also provide to the production of

adipokines.

The increase in macrophage infiltration due to the expansion of adipose tissue during weight gain has been suggested to originate from pre-adipocytes or from bone marrow precursors [101], while other data indicate leptin to be responsible for macrophage diapedesis into white adipose tissue (WAT). The adipocyte is also able to secrete MCP-1, a chemokine overexpressed in obesity, which is responsible for recruiting circulating

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monocytes [102]. With adipocyte expansion and hypertrophy and subsequent local hypoxia, several pro-inflammatory adipokines are upregulated.

1.3.2 CRP

CRP is an acute-phase protein and a facilitator of innate immunity by binding to microbial polysaccharides and ligands on damaged cells activating the complement system, eventually causing the uptake by phagocytic cells [103]. This acute-phase protein has a hepatic origin and ensues a rise in interleukin-6 (IL-6) produced by macrophages and adipocytes [104], it is also stimulated by cytokines TNF-α and interleukin-1β (IL-1β) following an acute-phase inflammatory response [105]. With a relatively short half-life of ∼19 h, CRP levels decrease with a cease in inflammation.

Among healthy normal weight individuals, average CRP levels are <2 mg/l, while obese men and women are between 2-6 times more likely to have elevated CRP levels [106].

Existing research indicate CRP concentrations of 3-10 mg/l predict an increase risk of MI and stroke [107]. CRP levels of 10-40 mg/l are commonly found during mild

inflammation or viral infections [108]. During illnesses such as sepsis, CRP can increase to 300 mg/l [109]. CRP can be measured as serum CRP (S-CRP) or high sensitive CRP (hs-CRP), hs-CRP measures the same protein but has a lower detection range of 0.05 mg/l as opposed to 0.6 mg/l for S-CRP.

CVD is associated with elevated markers of systemic inflammation; studies have

established CRP as a strong and sensitive risk predictor of future cardiovascular events in healthy men and women [110, 111]. Specific CRP cut-off values have also been proposed according to level of CVD risk, CRP <1 mg/l is associated with low risk while CRP between 1.0-3.0 mg/l represent an intermediate risk and CRP levels over 3.0 has a high risk [112].

According to the European Society of Cardiology guidelines from 2007, CRP was not recommended for prediction of cardiovascular risk in routine practice [113]. Since then, there has been some debate regarding the value of measuring levels of CRP and other biomarkers of inflammation for risk prediction in CVD. In a recent analysis of 52 cohort

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CVD risk among people without known CVD, found that adding CRP could prevent one additional cardiovascular event over a period of 10 years for every 400-500 subjects screened [114]. However, the CANTOS study published in 2017, showed a strong

association in reduction in hs-CRP, in thes absence of LDL-C change, with cardiovascular event and all-cause mortality reduction following canakiumab therapy [115].

1.3.3 Weight loss and CRP

As adipose tissue play a role in the production and regulation of inflammatory cytokines, such as IL-6 that stimulate CRP production, it has been suggested that weight loss may be one of the ways by which inflammation is reduced and therefore also why we see a reduction in CVD risk [116]. Several studies suggest that weight loss could lead to a reduction in CRP levels [117] [118]. Accordingly, a reduction in body weight is a probable cause for reduced CRP levels.

1.3.4 Dietary fat and inflammation

A variety of dietary factors enhance or reduce inflammation [119]. The current nutrition recommendations for fats give information on both the total amount as well as types of fats a healthy dietary pattern should consist of [61]. Fat is not only a concentrated energy source, but dietary fats also provide us with essential fatty acids and fat-soluble vitamins, today’s total fat recommendation therefore ranges somewhere between 25-40 E%. Although a high total fat intake has been associated with an increase in obesity [120, 121], and a following sub-clinical inflammatory state, types of dietary fat can affect the inflammatory response [122, 123].

Saturated fatty acids

Several studies have found a positive association between a high saturated fat intake and inflammation [124] [125]. Lopez-Garzia et al. found a positive link between a Western dietary pattern and CRP levels, although the study focused on dietary patterns and not on saturated fat as an individual nutrient, the overall dietary composition of the Western dietary pattern was high in trans and SFA [125]. King and associates studied the

NHANES data from 99-00 and found a moderate association with saturated fat

consumption and increased CRP levels [124]. The SYSDIET study observed an elevation

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of Interleukin-1 Receptor antagonist (IL-1 RA), a sensitive marker of inflammation in obesity, with an increase in SFA intake, CRP levels was however unchanged [90].

Figure 1. Dietary FFA modulation of immune response

Published with permission from Oxford Press (License number: 4234581247639)

Infection of cells by microorganism activates an innate immune response mediated by pattern recognition receptors (PRRs), which include Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain proteins (NODs) among others. These PRRs activate downstream signaling pathways, which lead to the expression of inflammatory mediators [126]. Research has demonstrated that PRRs can be controlled by specific dietary fatty acids, thereby differentiating between SFA and PUFA. From this argument, SFA may enhance PRR activation and foster a pro-inflammatory state by up regulating toll-like receptor 4 (TLR4), who’s increased expression has been reported in

atherosclerotic tissues. While SFA such as lauric and palmitic acid can activate TLR2 and TLR4, PUFA inhibit this activation [122].

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Polyunsaturated fatty acids

Most healthy dietary patterns emphasize intake of LA and alpha linoleic acid (ALA), owing to PUFAs’ favorable effect on CVD through an improvement of the lipid profile and its role in inflammation. Current dietary guidelines recommend a PUFA intake between 5-10E%, where ω-3 fatty acids should contribute at least 1 E%. The connection between dietary PUFA, inflammation and disease is to some degree linked to changes in the PUFA content of phospholipids in cells such as monocytes, macrophages, and vascular

endothelial cells, where the PUFA content affect inflammation through membrane fluidity and receptor function among others [127].

Eicosanoids, generated from PUFAs are important regulators of inflammation, with arachidonic acid AA as the key substrate for eicosanoid synthesis. The eicosanoids;

prostaglandins (PGs), thomboxanes (TX), leukotrienes (LTs) are generated from AA and regulate the intensity and duration of inflammation [128]. Especially the ω-3/ ω-6 ratio has been identified as a potential factor in the pathogenesis of this inflammatory

response, due to ω-3 and ω-6 fatty acid competition in uptake and metabolism [129], thereby causing ω-6 fatty acids attenuating the beneficial effects of ω-3 fatty acids.

In a cross sectional study of the Health Professionals follow-up and Nurses Health II study dietary LA intake was not associated with increased levels of CRP, IL-6 or soluble tumor necrosis factor 1 and 2. The inverse relationship found between ω-3 fatty acids from fish and soluble TNF receptor levels were actually stronger for higher ω-6 fatty acid intake [130]. Other studies have also found an inverse relationship between LA intake and inflammatory markers [131, 132]. A systematic review of RCTs from 2012 concluded that there is no evidence to support an increase in inflammation from dietary LA intake [133].

Fatty fish and fish oils contain high proportions of EPA and DHA. Both animal and human studies show that consuming these food items results in increased amounts of EPA and DHA in the phospholipids of the inflammatory cells, mainly at the expense of AA. Animal studies also show that fish-oil is significantly more effective than ALA in altering

eicosanoid metabolism and phospholipid cell content [134]. A reason could be that although ALA is converted to EPA and DPA, only limited amounts are converted to DHA

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[135]. Several clinical trials have shown a dietary pattern high in PUFA and ALA to reduce CRP levels [135-137].

1.3.5 Fiber

Numerous cohort studies have examined the effect of fiber on CVD, and have found a positive inverse relationship [138]. Mechanism such as lowering of cholesterol, BP, and regulation of glucose and body weight has been suggested. However, another possible mechanism is through regulation of the inflammatory response. Intake of dietary fiber was shown to be inversely associated with CRP levels in the NHANES 99-00 cross- sectional study [139]. Data from the Women’s Health Initiative Observational Study found a high-fiber dietary pattern to be associated with lower levels of IL-6 and TNF α- R2, but this link was not established between fiber and CRP [140]. A small randomized crossover study found a fiber intake of about 30g/day either naturally in a dietary pattern or from supplements to reduce CRP levels. Although absolute changes were similar across normal and obese weight subjects, the change was not significant in the obese hypertensive participants, most likely due to higher baseline values [141].

No certain mechanism has been established as for how fiber potentially modulate the inflammatory response, but possibilities such as slowing the absorption of glucose, modulation of oxidative stress and a change in gut flora have been suggested [141].

1.4 Glucose metabolism and fatty acids

CVD is the most common cause of morbidity and mortality among men and women with diabetes, accounting for roughly 50-70% of deaths [142, 143]. Men with diabetes have about a 2-fold increased risk of CVD while women have close to a 4-fold increase

compared to their respective healthy counterparts [143]. Data also suggest that subjects with diabetes without history of CVD have an equal risk as of having a myocardial

infarction (MI) as subjects with history of previous CVD[144]. Although an association between the level of glycemic control and CVD development have been shown in

epidemiological studies, it is a weaker link compared to the association of hypertension and dyslipidemia and CVD [145]. However, as epidemiological studies only show an

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1.4.1 HbA

1

c and fasting plasma glucose

HbA1c refers to glycated hemoglobin, a form of hemoglobin that is measured to establish the average plasma glucose levels for the past 6-8 weeks. It can be measured any time of the day as fasting is not needed, and is the preferred test for evaluating glycemic control primarily in people with diabetes, but can also be used as a diagnostic tool to asses risk for developing diabetes. According to the American Diabetes Association and the

Norwegian guidelines having an HbA1c >6.5% is categorized as diabetes, and 5.7-6.4% as pre-diabetes. Fasting plasma glucose levels on the other hand is measured after at least 8 hours of fasting and levels equal to or above 7 mmol/l is defined as diabetic [146].

1.4.2 Insulin resistance

Insulin resistance is defined as a condition where liver, skeletal muscle and adipose cells become less sensitive to normal levels of insulin, a hormone produced by the beta cells in the pancreas facilitating glucose absorption, metabolism and storage [147]. In the developing stages of insulin resistance, the beta cells in the pancreas responds by increasing insulin secretion in an effort to compensate and keep blood glucose levels within range. In obesity and T2DM, insulin resistance in adipose tissue and skeletal muscle is expressed through decreased insulin-stimulated glucose transport and metabolism and diminished suppression of gluconeogenesis, glycogenolysis from the liver

1.4.3 FFA and insulin resistance

Both animal and human studies have revealed that excessive intake of fat produces insulin resistance. Elevated plasma free fatty acids (FFA) can be due to ingested fat, increased lipolysis from fat stores and also decreased FFA clearance [148]. Acute elevation of FFA in the blood causes insulin resistance, this response mainly takes place in skeletal muscle where >80% of insulin stimulated glucose uptake occurs [149]. This mechanism is not gender specific and also apparent in subjects without diabetes, and persists in long-term elevations of plasma FFA [150, 151]. Most of the endogenous glucose production takes place in the liver, this process is increased in T2DM patients with reduced adherence to medical advice, and correlates with elevated fasting plasma

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glucose levels. Researchers have suggested that the increase in endogenous glucose production can be linked to FFA’s inhibition of insulin suppression of hepatic glucose production, acutely through insulin inhibiting glycogenolysis and later also due to an increase in gluconeogenesis [149].

FFA in plasma enters cells to either be oxidized to generate ATP or re-esterified to TG and stored. The increase in plasma FFA results in a buildup of metabolites implicated in FFA re-esterification, involving long chain acyl-CoA and diacylglycerol (DAG), which activates protein kinase C (PKC) and other serine/threonine kinases. The exact mechanism of how these kinases are activated is somewhat unclear but may include glycogenolysis and lipolysis. Thus FFA induced insulin resistance results in reduced glucose uptake in muscle and increased glucose production in the liver, in the end causing hyperglycemia [151].

Figure 2. Potential mechanism of FFA on insulin resistance

Permission requested from Oxford Press

1.4.4 PUFA in glucose metabolism

Initial research on fat and the glucose metabolism failed to consider that different fatty acids could exert different effects. Since then studies have found PUFA, especially ω-3 fatty acids from fish and fish oils to be beneficial.

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In the liver ω-3 fatty acids cause the activation of peroxisome proliferator-activated receptor alpha (PPARα), which stimulates the formation of peroxisomes and a resulting increase in fatty acid oxidation in peroxisomes over mitochondrial β-oxidation.

However, this effect has been proven more effective and beneficial in animal studies. ω-3 fatty acids may also activate AMP-protein kinase (AMPK) and regulate the activity of the transcription factors PPARα and sterol regulatory element binding protein 1 (SREBP-1) and as a result triacylglycerol (TG) in liver decreases and we se a following increase in hepatocyte insulin sensitivity.

In adipose tissue ω-3 fatty acids modulate gene expression through transcription factors, where members of the PPAR-family are important nuclear receptors. Over-expression of PPARγ in WAT in mice was found to improve insulin sensitivity, hence illustrating the importance of WAT in glucose metabolism [152]. The effect of PPARγ could similarly extend to muscle cells by increasing insulin sensitivity. ω-3 fatty acids also affect the expression of the glucose transporter type 4 (GLUT-4) gene and hence glucose transport into the fat cells [153], in animal studies it has been suggested that PUFA enrich muscle plasma membranes and improve glucose uptake and insulin sensitivity by lengthening the amount of time the glucose transporter-4 is associated with the membrane [154].

Nevertheless, although animal studies are insightful to researching mechanisms they are not always transferable to human trials. A rodent study looking at if supplemented fish oil could reverse the negative metabolic effects of a high fructose dietary pattern found an attenuated homeostasis model assessment -insulin resistance (HOMA-IR)-index response those receiving 5 E% and 7E% from fish oil [155]. The current

recommendation according to the NNR12 is at least 1E%, thus an intake of 5-7E% is most likely not approachable at a population level.

In a recent RCT the effects of dietary ω-3 fatty acids (3.9g/day) on insulin sensitivity in non diabetic, obese and insulin resistant humans was compared using placebo and ω-3 fatty acid supplementation. The study found no effect on insulin sensitivity as well as no change in postprandial glucose levels [156], another study following a similar

intervention found an improved insulin sensitivity response, but changes were not significant compared to the control group [157].

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Numerous studies have looked at the effect of a Mediterranean dietary pattern on CVD risk factors. The largest and most discussed is the PREDIMED trial, which compared the effect of two Mediterranean dietary patterns, either supplemented with nuts or extra virgin olive oil (EVOO), with a control dietary pattern. Not only did they find a beneficial effect on BP and cholesterol but plasma glucose levels were also significantly improved in both intervention groups [158]. A cross sectional study on diabetic patients, observed an inverse trend with high adherence to a Mediterranean dietary pattern with HbA1cand post prandial glucose levels, as well as a significant decrease in the respective

parameters [159]. The high MUFA and PUFA content of the Mediterranean dietary appear to be responsible for the observed health benefits.

A systematic review from 2016, discovered 5E% carbohydrate exchanged with SFA, MUFA, or PUFA did not significantly alter fasting glucose levels. However, replacing SFA with PUFA was linked to a significant decrease. The same review also found replacing 5E% from carbohydrate or SFA with 5E% from either MUFA or PUFA, each lowered HbA1csignificantly (p<0.001) [160]. Similar trends were also seen for HOMA-IR and insulin levels. In conclusion it appears that PUFA has the most beneficial effects. MUFA except for lowering HbA1c and improving HOMA-IR in comparison to both carbohydrate and SFA, has no effect on blood glucose levels. Thus is seems although ω-3 fatty acids has proven an effect in animal studies, ω-6 fatty acids show the most promising result in humans with regards to glucose metabolism.

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2 Study objectives

The main objective of this thesis is to study if the effects of PUFA and SFA on metabolic risk factors for CVD differ between normal weight and obese individuals with elevated LDL-C (>3.0mmol/l).

2.1 Specific objectives

- To investigate if there are differences in how normal and obese individuals respond to a PUFA or SFA dietary pattern by assessing the following parameters

o SBP and DBP o CRP

o Glucose and HbA1c

2.2 Hypothesis

Normal weight and obese subjects with elevated LDL-C respond differently to a SFA or PUFA dietary pattern with regards to the following CVD risk factors; SBP, DBP, CRP levels, glucose and HbA1c

H0: SBP, DBP, CRP levels, glucose and HbA1c will not differ between normal weight and obese individuals when substituting saturated fat for unsaturated fat

H1: SBP, DBP, CRP levels, glucose and HbA1c will differ between normal weight and obese individuals when substituting saturated fat for unsaturated fat

The effect of saturated –and unsaturated fat on inflammation, blood pressure and glucose metabolism in normal -and obese weight men and women with elevated LDL-cholesterol: A randomized, clinical trial.