The Pennsylvania State University. The Graduate School. College of Health and Human Development

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1 The Pennsylvania State University The Graduate School College of Health and Human Development EFFECTS OF DIETS LOW IN SFA AND WITH VARYING MUFA AND PUFA PROFILES ON BODY COMPOSITION AND HDL FUNCTION IN INDIVIDUALS WITH OR AT RISK FOR METABOLIC SYNDROME A Dissertation in Nutritional Sciences by Xiaoran Liu 2015 Xiaoran Liu Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2015

2 The dissertation of Xiaoran Liu was reviewed and approved* by the following: Penny M. Kris-Etherton Distinguished Professor of Nutritional Sciences Dissertation Advisor Chair of Committee Sheila G. West Professor of Biobehavioral Health Michael H. Green Professor of Nutritional Sciences Joshua D. Lambert Associate Professor of Food Science Gordon Jensen Head of Department of Nutritional Sciences *Signatures are on file in the Graduate School

3 ABSTRACT Monounsaturated fatty acids (MUFA) decrease risk of metabolic syndrome (MetS). There is evidence that vegetable oils high in MUFA lower CVD risk in individuals with MetS. A multi-center, double blind, randomized, 5-period crossover, controlled feeding study was conducted. Subjects (n=101: 51 women, 50 men) were fed an isocaloric heart healthy diet containing one of the five treatment oils incorporated in a smoothie for 4 weeks followed by a 4 weeks break between experimental diets. The five vegetable oils were: Canola oil (62.8% MUFA, 29.3% PUFA: 19.5% LA, 10% ALA), CanolaOleic oil (72% MUFA, 17% PUFA: 15% LA, 2% ALA), CanolaDHA (63.8% MUFA, 13% LA, 6% DHA), corn/safflower oil (69.3% LA, 17.6% MUFA) and flax/safflower oil (69.4% PUFA: 37.5% LA, 32% ALA, 17.9% MUFA). The first study was designed to evaluate the efficacy of five vegetable oils (low in saturated fatty acid (SFA) with varying unsaturated fatty acid profiles) on abdominal fat mass in subjects with or at risk for MetS. After four weeks, reductions in abdominal fat mass were observed when the Canola ( g, p=0.0264) and CanolaOleic oil diets ( g, p=0.0245) were compared with the Flax/Saff oil diet. The reduction of android fat mass from baseline in response to the Canola (p=0.042) and CanolaOleic (p=0.007) were both significant. There was no difference in changes of abdominal fat mass from baseline for the CanolaDHA, Corn/Saff and Flax/Saff oil diets. The android to gynoid fat mass ratio was decreased in males after the CanolaOleic oil diet compared with the Flax/Saff oil diet (0.71 versus 0.73, p=0.0067). Attenuation of central obesity was associated with a reduction in blood pressure in response to the Canola (SBP r = 0.26, p=0.062; DBP r=0.38, p=0.0049) and CanolaOleic oil diets (SBP r = iii

4 0.39 p=0.004; DBP r=0.45, p=0.0006). Moreover, the decrease in central obesity was associated with a reduction in TG on the CanolaOleic oil diet (r = 0.42, p=0.0017). The second study was designed to investigate the effects of five vegetable oils on HDL function measured via cholesterol efflux capacity. The Canola, CanolaOleic, CanolaDHA, Corn/Saff and Flax/Saff oil diets increased serum mediated CEx capacity from THP-1 macrophages by 39.1%, 33.6%, 55.3%, 49.2% and 50.7% respectively, compared to baseline (p<0.05 for all). Weight status was an independent predictor of the serum mediated CEx capacity. Participants with a normal BMI had a greater increase in CEx capacity compared with overweight and obese participants. Waist circumference and abdominal adiposity were negatively correlated with serum mediated CEx capacity (r = -0.25, p = 0.012, r = -0.33, p = 0.017, respectively). The third study was designed to explore the relationship among VAT, SAT and anthropometric measurements in a subgroup of study participants (n=14). Regression analysis demonstrated that at baseline, in both males and females, CRP was positively correlated with VAT mass (r = 0.92, p < ). Waist circumference was positively correlated with SAT mass (r = 0.59, p = 0.034). In addition, BMI was positively correlated with SAT mass (r = 0.83, p = ). There were no correlations for CRP level and SAT mass; waist circumference and VAT mass; and BMI and VAT mass. Overall, the results showed that two of high MUFA diets (Canola and CanolaOleic) had beneficial effects on reducing abdominal fat mass and improving MetS risk in individuals with or at risk for MetS. All diets low in SFA increased CEx capacity indicating an iv

5 improvement in HDL functionality. Waist circumference was positively correlated with SAT but not VAT. VAT mass was associated with increased inflammation status. v

6 TABLE OF CONTENTS List of Figures... ix List of Tables....x Abbreviations... xi Acknowledgements... xii Chapter INTRODUCTION Background Premise of this Dissertation Conclusion... 2 Chapter LITERATURE REVIEW Metabolic Syndrome: Definition and Prevalence Definition Prevalence Effect of MUFA on MetS Criteria Effects of Monounsaturated Fatty Acids on Weight Management and Central Obesity Effects of Monounsaturated Fatty Acids on Triglyceride and HDL-C Effects of Monounsaturated Fatty Acids on Glycemic Control Effects of Monounsaturated Fatty Acids on Blood Pressure Summary Objectives and Hypothesis Chapter CANOLA AND HIGH-OLEIC ACID CANOLA OILS REDUCE ABDOMINAL FAT MASS IN INDIVIDUALS WITH CENTRAL ADIPOSITY Abstract Introduction Methods Participant Characteristics Experimental Diets Dual-Energy X-Ray Absorptiometry (DXA) measurements Plasma Fatty Acids Statistics Results vi

7 3.5.1 Participant Characteristics Diet-specific Effects on the Plasma Fatty Acid Profile Diet-specific Effects on Body Weight and Body Composition Diet-specific Effects on Changes in Body Weight and Body Composition from Baseline Correlation between Android Fat Mass Changes and Cardiometabolic Risk Factors in Response to Diet Sub-analysis Between Genders and Plasma Fatty Acid Levels Discussion Conclusion Supplemental Material Chapter LITERATURE REVIEW Introduction The HDL-C Hypothesis Controversy HDL and Reverse Cholesterol Transport Effect of Dietary Bioactive Compounds on HDL-C Levels Effect of Dietary Bioactive Compounds Cholesterol Efflux Summary Objectives and Hypothesis Chapter VEGETABLE OILS WITH DIFFERENT UNSATURATED FATTY ACID PROFILES INCREASE SERUM MEDIATED CHOLESTEROL EFFLUX FROM THP-1 MACROPHAGES: THE CANOLA OIL MULTICENTRE INTERVENTION TRIAL Abstract Introduction Methods Study Design Sample Collection Dual-Energy X-Ray Absorptiometry (DXA) Measurements High Throughput Screen Cholesterol Efflux Assay Statistical Analysis Results Baseline Characteristics Diet-Specific Effects on the Lipid/Lipoprotein Profile Diet-Specific Effects on Serum Mediated Cholesterol Efflux from THP-1 Macrophages Correlation Between Waist Circumference, Abdominal Fat Mass and Serum Mediated Cholesterol Efflux at Baseline Discussion Conclusion vii

8 Chapter MEASUREMENT OF ABDOMINAL ADIPOSE TISSUE IN A SUBSET OF COMIT PARTICIPANTS: A COMPARISON of MRI, DXA and ANTHROPOMETRIC MEASUREMENT Abstract Introduction Materials and Methods Anthropometric Measurements DXA Measurements MRI Adipose Tissue Quantification Statistical Methods Results Participant Characteristics Correlations between VAT, SAT Mass and Anthropometric Measurements Discussion Chapter SUMMARY, LIMITATIONS AND FUTURE DIRECTIONS Appendix 7-day Cycle Menus for COMIT REFERENCES viii

9 LIST OF FIGURES Figure 3-1. Study Design for COMIT Figure 3-2. Recruitment Flowchart Figure 3-3. Body weight android fat mass and trunk fat mass changes in response to five experimental diets (n=54; males n=20, females n=34) Figure 3-4. Total lean mass and gynoid lean mass changes in response to five experimental diets (n=54; males n=20, females n=34) Figure 4-1. Reverse cholesterol transport Source (116) Figure 4-2. Illustration of LXR regulatory cholesterol efflux pathway from macrophages Figure 5-1. Changes in the CEx capacity in response to the test diets Figure 5-2. Changes in the CEx capacity in response to BMI status Figure 5-3. Correlation between waist circumference and CEx capacity at baseline for all participants (n=101) Figure 5-4. Correlation between abdominal fat mass and CEx capacity at baseline in a subgroup of participants (n=54) Figure 6-1. Axial slices selection Figure 6-2. Segmentation method Figure 6-3. A) Correlation between abdominal fat mass and VAT; B) Correlation between abdominal fat mass and SAT Figure 6-4. A) Correlation between WC and VAT mass; B) Correlation between WC and SAT mass Figure 6-5. Correlation between MRI obtained and DXA obtained VAT mass ix

10 LIST OF TABLES Table 3-1. Fatty acid profiles of the five treatment oils Table 3-2. Energy and nutrient profiles of the experimental diets Table 3-3. Metabolic characteristics of participants at screening Table 3-4. Body composition of participants at baseline Table 3-5. Correlation between adipose tissue mass and cardiovascular risk factors at baseline (n=54) Table 3-6. Endpoint body weight and body composition after each of the experimental diets (n=101; males n=50, females n=51) Table 3-7. Correlation between android fat mass changes and changes in cardiovascular risk factors (n=54) Table Plasma fatty acid profiles in response to the experimental diets Table Plasma fatty acids as predictors of android fat mass changes in response to Canola, CanolaOleic, CanolaDHA oil diets Table 4-1. Pathways for macrophage-specific cholesterol efflux Table 4-2. Effects of bioactive compounds on HDL-C levels in randomized controlled Trials Table 4-3. Effects of bioactive compounds on macrophage and HDL-mediated cholesterol efflux Table 5-1. Baseline anthropometric and cardiometabolic characteristics Table 5-2. Body composition of participants at baseline Table 6-1. Baseline anthropometric and cardiometabolic characteristics Table 6-2. Baseline body composition Table 6-3. Spearman correlations of baseline SAT, VAT and body composition measurements x

11 ABBREVIATIONS CE CEx CHD CHO COMIT CRP CVD DBP DXA EE FA HDL-C LDL-C MetS MRI MUFA PPAR PUFA RCT SAT SBP SFA TC TE TG T2D VAT WC Cholesterol Ester Cholesterol Efflux Coronary Heart Disease Carbohydrate Canola Oil Multicentre Intervention Trial C-Reactive Protein Cardiovascular Disease Diastolic Blood Pressure Dual-energy X-ray Absorptiometry Energy Expenditure Fatty Acids High-Density Lipoprotein Cholesterol Low-Density Lipoprotein Cholesterol Metabolic Syndrome Magnetic Resonance Imaging Monounsaturated Fatty Acids Peroxisome Proliferator-Activated Receptor Polyunsaturated Fatty Acids Randomized Controlled Trial Subcutaneous Adipose Tissue Systolic Blood Pressure Saturated Fatty Acids Total Cholesterol Total Energy Triglyceride Type 2 Diabetes Visceral Adipose Tissue Waist Circumference xi

12 ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my advisor and committee chair, Dr. Penny Kris-Etherton for her guidance and support. I am very much appreciative for all of the opportunities she has provided that significantly enhanced my research experience. She encouraged me to express my thoughts and challenged me with new tasks. Her passion for science is admirable and motivating. I am forever grateful for her mentorship. I would like to thank my co-advisor, Dr. Sheila West, who has taught me so much in statistical analysis and SAS coding. Her encouragement and support are very much appreciated. Dr. Michael Green who challenged me with the Max question has ignited my interests in lipids research. I really appreciate his efforts on guiding me through the process. As Dr. Green suggested, I will always work towards the light bulb moment in my future research. I would like to thank Dr. Lambert for his support, expertise and excellent advice. He always reminds me of the food aspect of nutrition. I would like to thank the COMIT research team. It was my privilege to work with Dr. Peter Jones, Dr. David Jenkins, Dr. Benoît Lamarche and their research teams. I have learned so much from every single one of them. I believe such experiences will benefit me greatly in my future career in research. This page is not long enough to list everyone who has generously lent me a hand through this journey. This clinical study could not have been completed without clinical staff from Dr. Kris-Etherton s lab and Clinical Diet Center and nursing staff at the Clinical Research Center. I really appreciate Jennifer Fleming, Marcella Smith for their great assistance xii

13 through the COMIT trial. I would like to thank Tracey Banks who has helped me greatly during the study and has taught me so much about dealing with difficult situations in life. Finally, I want to give special thanks to my family, Yang Liu, Meiying He, Miaojian Zhang, Yabing Guo and Haitao Zhang for their love, support and encouragement. I greatly appreciated Mr. Miaojian Zhang s years of support and love from Manitoba, Canada to Pennsylvania; he is the person who always has faith in me. For that, I thank you. xiii

14 Chapter 1 INTRODUCTION 1.1 Background The growing prevalence of obesity is a worldwide public health concern. In U.S., more than 60% of population is overweight or obese. Obesity is associated with many health concerns including cancer, cardiovascular disease, dyslipidemia and T2D (1, 2). Obesity related metabolic disorders can be prevented through lifestyle and/or diet interventions. The AHA/ACC/TOS Guideline for the Management of Overweight and Obesity in Adults recommends a healthy dietary pattern that can vary in macronutrient profiles (3). The Dietary Guidelines for Americans 2010 recommended consuming less than 10% of calories from SFA by replacing them with MUFA and PUFA (4). Vegetable oils are important sources of MUFAs and PUFAs. Evidence from clinical trials have shown that replacing SFA or Trans-fatty acids with MUFA or PUFA improves blood lipid profiles thus reducing CVD risk (4). Emerging evidence demonstrates that dietary MUFA has beneficial effects on reducing CVD risk. However, few studies have investigated the effects of consuming MUFA with respect to body weight management, body composition changes and HDL functionality. Research addressing these questions will provide insights about modifying macronutrient profiles to increase MUFA intake as a strategy for weight management in overweight and obesity. 1

15 1.2 Premise of this Dissertation The premise of this dissertation was to investigate the effects of diets containing vegetable oils with varying n-9, n-6 and short and long-chain n-3 fatty acid profiles on body composition changes, cardiometabolic risk, HDL functionality, and adipose tissue distribution in individuals at risk or with MetS. The present study was a multi-center, double blind, 5-period crossover controlled feeding trial. These results of the thesis add to the evidence base that dietary MUFA can be a strategy for preventing (and treating) MetS. 1.3 Conclusion The study presented in this dissertation contributes to the knowledge of diets low in SFA and high in MUFA can favorably modify body composition. In addition, diets low in SFA and high in unsaturated fatty acids (including MUFA and PUFA) improve HDL function. The following chapter reviews the literature of the MUFA studies and their effects on weight management and MetS risk factors. In Chapter 3, the results of controlled feeding trials investigating the effects of five vegetable oils with low SFA and varying unsaturated fatty acid profiles on body weight and body composition are presented. Chapter 4 reviews the literature on effects of dietary bioactive compounds on HDL-C levels and HDL function. Chapter 5 presents the effect of 5 diets on HDL functionality as well as the relationship between HDL function and parameters of central obesity. Chapter 6 presents VAT, SAT mass assessed by two noninvasive body imaging methods and their association with anthropometric measurements in a subgroup of study 2

16 participants. Chapter 7 includes a research summary and discussion of future research directions. 3

17 Chapter 2 LITERATURE REVIEW 2.1 Metabolic Syndrome: Definition and Prevalence Definition Metabolic syndrome (MetS) is a growing public-health problem domestically and worldwide. MetS is a composite of risk factors including increased waist circumference, blood glucose, TG, blood pressure, and reduced high-density lipoprotein cholesterol (5, 6). Abdominal obesity is a key criterion of MetS. According to the International Diabetes Federation definition (IDF), for a person to be defined as having MetS, they must have central obesity (males 94 cm, females 80cm) plus any two of the following four factors including high TGs 150 mg/dl, low HDL-cholesterol (males< 40 mg/dl, females < 50 mg/dl), and high blood pressure (SBP 130 or DBP 85 mm Hg), and fasting plasma glucose 100 mg/dl (5) Prevalence MetS is associated with increased cariometabolic risk. The likelihood of developing CVD within 10 years is increased by 2-fold and diabetes by 5-fold in individuals with MetS (7). During the past decade the prevalence of MetS has increased substantially in the US; Reference to the National Health and Nutrition Examination Survey (NHANES) , almost 40% of adults in the US have MetS based on the IDF (8). From

18 to 2010, the age-adjusted prevalence of MetS decreased from 25.5% to 22.9% (1). Although the overall prevalence of MetS has declined, there is discrepancy in trends for individual MetS criteria. Specifically, the prevalence of elevated blood pressure has declined from 32.3% to 24.0% from 1999 to 2010 (p < 0.001). There is a similar trend for the prevalence of hypertriglyceridemia with estimates of elevated TGs in total population declining from 33.5% to 24.3% (p < 0.001). Also, there is a decline in the prevalence of low HDL-C from 38.5% to 30.1% (p < 0.001). It is noteworthy that concomitant with improvements in the prevalence of elevated blood pressure and dyslipidemia, usage of anti-hypertensive drugs and lipid modifying agents has also increased over the past two decades. The favorable trends in elevated blood pressure and dyslipidemia may reflect the effects of pharmacological intervention. In contrast, prevalence of hyperglycemia in the total population increased from 12.9% to 19.9%. As the key criterion of MetS, the prevalence of central obesity, measured by WC has increased from 45.4% to 56.1% over time. 2.2 Effect of MUFA on MetS Criteria There is accumulating evidence that MUFA reduce MetS criteria and, thereby, decrease risk of MetS. Dietary MUFA favorably modulate body weight, promote healthy blood lipid/lipoprotein profiles, blood pressure and improve insulin sensitivity. 5

19 2.2.1 Effects of Monounsaturated Fatty Acids on Weight Management and Central Obesity There is a perception that fat intake is associated with increased body weight leading to obesity and associated cardiometabolic risk. It has been proposed that the quality of dietary fat intake should be considered in addition to the quantity of dietary fat. Evidence from both epidemiological and clinical studies has shown that MUFA intake is not associated with increasing the risk of obesity. In contrast, emerging evidence has demonstrated that a higher MUFA intake benefits body weight management. Schwingshackle et al. evaluated 12 RCTs studies (6 months-24 months) that assessed the effects of body weight changes in response to high-mufa (>12% of kcal) versus low-mufa (<12% of kcal) diets (9). There was no significant difference between the high MUFA diet versus the low MUFA diet on body weight (-0.82kg, p=0.16). However, subsequent post-hoc analysis revealed that reduction of body weight was significantly more pronounced following a high-mufa diet (-1.71kg, p=0.05) when compared with a low fat diet (total fat content <30% kcal, SFA < 7-10% kcal). In four studies including body fat mass assessment, a significant reduction of -1.94kg fat mass was observed after consumption of the high-mufa diet when compared with the low- MUFA diet (10-13). The potential mechanisms that may explain the effects of MUFA consumption on body composition changes include increased fat oxidation and energy expenditure. Lovejoy et al. (14) examined the effects of a moderate fat diet (28% of total energy) with 9% of total energy from MUFA (oleic acid) or SFA (palmitic acid), or trans-fa (elaidic acid) on substrate oxidation. Each diet period was 4-wk with a 2-wk washout period in between. In twenty-five healthy men and women who have completed the study, resting 6

20 energy expenditure, substrate oxidation were measured in the fasting state as well as in the postprandial state following consumption of the test meals. There was no significant difference between MUFA, SFA and Trans-FA on energy expenditure and fat oxidation rate. In contrast, Piers et al. reported that a high MUFA diet induced weight loss and fat mass loss compared to a diet high in SFA (15). A randomized crossover study design was implemented to evaluate body composition changes in response to either a high MUFA diet (22% of total energy) or a high SFA diet (24% of total energy) in 8 overweight/obese male participants. After four weeks of consumption, the MUFA diet resulted in a greater weight loss (-2.1kg vs. 0.5kg, p=0.0015) compared to SFA-rich diet (15). Moreover, the same study observed a greater fat mass loss after the high MUFA diet compared to the high SFA diet (-1.1% vs. 0.9%, p=0.0034). In another study, Kien et al. reported a higher fat oxidation rate and greater EE after a 28 days consumption of a MUFA rich diet compared with a high SFA diet (16). Forty-three normal to overweight men and women consumed a high fat run-in diet (40% total energy as fat, with 13% as MUFA and 8% as SFA) for four weeks. Participants were then randomized to a high MUFA (n=22, 40% total energy as fat, 31.4% total energy as oleic acid) diet or a high SFA diet (n=21, 40% total energy as fat, 16.8% as palmitic acid, 16.4% as oleic acid). Each diet period was 28 days. At the end of the study, either a high MUFA or high SFA meal that corresponded to the intervention diet was fed to the participants. Fasting and postprandial energy expenditure and fat oxidation were measured. The high MUFA diet resulted in greater postprandial fat oxidation compared to the high SFA diet ( mg/kg fat free mass/min vs mg/kg fat free mass/min, 7

21 p=0.03). The daily EE in response to the high MUFA diet remained unchanged (9 kcal/day vs. -214kcal/day, p=0.02) but was decreased in the high SFA diet. This study adds to the evidence base that MUFA are more favorable compared to SFA with respect to weight maintenance. In addition, Schmidt et al. (24) reported that the fractional oxidation rate of oleic acid was significantly greater (21%) than palmitic acid in healthy men (n = 10) after consuming meals that provided 16.5% energy from oleate and 16.3% from palmitate every 20 minutes for 9 hours. Despite the supportive evidence about the beneficial effects of MUFA on body weight reduction and fat mass loss, there is limited information on the effects of MUFA on fat mass distribution. Paniagua et al. reported that compared to a carbohydrate-rich diet, a MUFA-rich diet resulted in greater fat oxidation rates, and a decrease in the abdomen-to-leg adipose tissue ratio in insulin resistant participants (17). Gillingham et al. reported a tendency for the android to gynoid fat mass ratio to decrease in hyperlipidemic participants after 28 days of consumption of high MUFA diet (18) Effects of Monounsaturated Fatty Acids on Triglyceride and HDL-C MUFA consumption has beneficial effects on improving the lipid/lipoprotein profile. In a meta-analysis of 9 short-term (2-6weeks) RCTs with weight-maintenance diets in T2D subjects, high MUFA diet ( 22% of TE) significantly improved lipids and lipoprotein profiles reducing TG by 19% and increasing HDL-C by 0.05 mmol/l when compared with a high CHO diet ( 49% TE, MUFA 7-13% TE) (19). In agreement with previous evidence, Kodama et al. reported in a meta-analysis including 11 intervention trials (crossover and parallel designs) with a duration of 10 days to 6 weeks that a MUFA 8

22 rich diet significantly decreased TG by 11% (p<0.001) compared with a low fat/high CHO diet in subjects with T2D (20). In comparison with a high CHO diet, the evidence favors a high MUFA diet having beneficial effects on reducing TG and maintaining HDL-C. Comparisons between high MUFA and low MUFA diets have been evaluated in clinical studies. Cao et al. conducted a meta-analysis to quantify the magnitude of changes in lipids and lipoproteins in response to a MF (moderate fat) blood cholesterol-lowering diet rich in unsaturated fat versus a LF (low fat) diet in subjects with and without diabetes (21). Among the 30 controlled-feeding studies with a total of 1213 subjects, the mean MUFA content of the MF diets was 23.6% (11.9% - 33% of TE) and 11.4% (6-15%) for the LF diets. In healthy participants, TG were significantly decreased and HDL-C was significantly increased in MF diet groups compared to LF diets. Similar results were reported in participants with diabetes for HDL-C and TG (21). There is a large evidence base comparing the effects of MUFA rich versus PUFA rich diets on the plasma lipid/lipoprotein profile (22-24). The evidence demonstrates that MUFA-rich diets have slightly less or similar TC lowering effects compared to the PUFA rich diets (25). When PUFA and MUFA rich diets were compared for replacement of dietary SFA in healthy adults, the MUFA rich diet maintains HDL-C levels to a greater extent with a 4% decrease in HDL-C levels compared to PUFA rich diets which decrease HDL-C levels by about 14% (22, 26) Effects of Monounsaturated Fatty Acids on Glycemic Control The epidemic of T2D has increased worldwide. An early meta-analysis of 10 randomized crossover trials conducted by Garg et al. compared isoenergetic high-mufa 9

23 (>22% of total energy) and high-cho (49-60% TE from CHO, 8-13% TE from MUFA) diets in patients with type 2 diabetes (19). An overall improvement in glycemic control and the lipid/lipoprotein profile in individuals with DM-1 or DM-II was reported when compared to high CHO diets (19). In a recent meta-analysis Schwingshackle et al. examined the effects of a high MUFA (>12%) diet versus low MUFA diet ( 12%) on glycemic control (27). Nine randomized controlled trials (after a minimum of 6 months of diet intervention) were included in this review. A beneficial Hb1Ac lowering-effect has been shown after consumption of a high MUFA diet compared with a low MUFA diet (27). Following these studies, the PREDIMED trial investigated the effects of long term adherence to Mediterranean diets supplemented with either virgin olive oil or nuts on individuals at high CVD risk compared to a lower-fat control diet. Three diets were randomly assigned to 418 non-diabetic subjects. After an average follow-up of four years, long term consumption of a high MUFA diet provided by extra virgin olive oil and mixed nuts reduced diabetes incidence by 52% when the two Mediterranean diet groups were pooled and compared with the control group. Evidence from recent clinical trials has shown beneficial effects of replacing SFA with MUFA in improving insulin sensitivity and glycemic control in both healthy individuals and individuals with/or at risk for T2D (28-30) Effects of Monounsaturated Fatty Acids on Blood Pressure Blood pressure is one of the most prevalent MetS risk factors. Evidence from clinical studies has shown that dietary MUFA either have neutral or hypotensive effects 10

24 when compared to diets rich in CHO, or n-3 and n-6 PUFA (31-33). Consistent hypotensive effects have been reported when MUFA are compared to a SFA rich diet (15). A meta-analysis including 10 intervention studies compared CHO ( 49% TE) and MUFA ( 19% TE) rich diets on blood pressure (34). MUFA rich diets were associated with a reduction In SBP (-2.6mmHg, p=0.02) and DBP (-1.8mmHg, p=0.05) compared with CHO-rich diets (34). Muzio et al. reported that after 5 months of consumption, a diet with 21% energy from MUFA was associated with a greater reduction in SBP compared to a diet high in CHO (65% TE from CHO, 14% TE from MUFA) in 100 obese subjects with MetS (33). In the Omni Heart Trial, the effect of diets with varying macronutrient profiles on blood pressure was assessed in 164 prehypertension or stage-1 hypertension subjects (32). After consumption of a diet with 21% of TE from MUFA, SBP (-1.3 mmhg, p=0.005) was significantly reduced compared to a CHO rich diet (58% TE from CHO) (32).. A recent meta-analysis including 12 dietary intervention studies investigated the long-term ( 6 months) effects of high-mufa (>12% total energy) vs. low-mufa ( 12% total energy) diets on cardiovascular risk factors and concluded that the high-mufa diet decreased SBP significantly compared with low-mufa diet by mmhg (p=0.03) in overweight/obese subjects (9). In addition, a high-mufa diet reduced the DBP by 1.15 mmhg (p=0.005) which is both clinically and statistically significant (9). The PREDIMED trial with 1224 high CVD risk patients has shown that participants with long term adherence to two high MUFA diets had significantly reduced DBP (-1.53 mmhg for the Mediterranean diet supplemented with extra virgin olive oil and mmhg for the 11

25 Mediterranean diet supplemented with mixed nuts) than the participants on the lower fat diet (35). The hypotensive effects of a MUFA rich diet were compared with a PUFA rich diet. In hypertensive participants, consumption of MUFA rich diet (17% TE from MUFA) for 6 months resulted in a 8 mmhg reduction in SBP (p=0.05), and a 6 mmhg reduction in DBP (p=0.01) compared to the PUFA rich diet (11% TE from PUFA) (31). Similarly, in 16 patients with T2D, a diet that provided 30% of energy as MUFA reduced 24-h SBP by 5 mmhg (p=0.02) and DBP by 3 mmhg (p=0.02) compared to a isoenergetic diet with 30% TE as PUFA (36). 2.4 Summary As dietary intervention remains the primary strategy for the prevention and treatment of MetS, recommendations for the optimal fatty acid profile remain to be clarified. Evidence favors dietary MUFA as a strategy to prevent or ameliorate MetS and CVD risk by improving the lipid/lipoprotein profile, blood pressure and insulin sensitivity. Dietary replacement of SFA by MUFA maintains HDL-C levels, reduces TG and improves glycemic control when compared with dietary CHO and PUFA. MUFA consumption favors body weight management. However, few studies have investigated the effects of dietary MUFA on central obesity. As one of the key criterion for MetS, central obesity is associated with increased cardiometabolic risk. There is a knowledge gap in our understanding of the effects of MUFA consumption in the regulation of fat mass deposition in the abdominal region. 12

26 2.3 Objectives and Hypothesis To examine of the effects of dietary oils low in SFA and high in MUFA and PUFA on body composition changes in individuals with or at risk for MetS. We hypothesize that diets high in MUFA will beneficially affect body composition via a decrease in total and/or regional fat mass and improve cardiometabolic risk. 13

27 Chapter 3 CANOLA AND HIGH-OLEIC ACID CANOLA OILS REDUCE ABDOMINAL FAT MASS IN INDIVIDUALS WITH CENTRAL ADIPOSITY 3.1 Abstract Background: A high MUFA diet has been shown to favorably affect metabolic syndrome (MetS) criteria, and emerging evidence suggests that high-mufa diet also decreases body weight. Whether a high MUFA diet reduces visceral adipose tissue mass, a central feature of MetS, however, is unclear. Objective: The objective of this study was to examine the effects of isocaloric diets containing vegetable oils with varying n-9, n-6 and longer-chain n-3 fatty acid profiles on body weight and body composition changes in a population with or at risk for MetS. Design: A multi-center, double blind, randomized, 5-period crossover controlled feeding study was conducted. Each treatment period lasted 4 wk and was followed by a 4-wk washout. Participants (n=101) with central obesity and at least one other MetS criterion consumed five isocaloric heart healthy diets (50% CHO, 15% PRO, 35% FAT, <7% SFA) containing different treatment oils (60g/3000kcal). Treatment oils were: Canola (62.8% 14

28 MUFA), CanolaOleic (72% MUFA), CanolaDHA (high-oleic canola oil with DHA, 63.8% MUFA with 5.8% DHA), Corn/Saff (17.7% MUFA, 69.3% LA), and Flax/Saff (17.9% MUFA, 37.5% LA, 32% ALA). Body composition was measured by dual-energy X-ray absorptiometry (DXA) at the beginning of the study and the end of each diet period. Results: After four weeks, reductions in abdominal fat mass were observed when the Canola (-54.59g, p=0.0264) and CanolaOleic oil diets (-66.09g, p=0.0245) were compared with the Flax/Saff oil diet. The reduction of android fat mass from baseline in response to the Canola (p=0.042) and CanolaOleic (p=0.007) were both significant. There was no difference in changes of abdominal fat mass from baseline for the CanolaDHA, Corn/Saff and Flax/Saff oil diets. The android to gynoid fat mass ratio was decreased in males after the CanolaOleic oil diet compared with the Flax/Saff oil diet (0.71 versus 0.73, p=0.0067). Attenuation of central obesity was associated with a reduction in blood pressure in response to the Canola (SBP r = 0.26, p=0.062; DBP r=0.38, p=0.0049) and CanolaOleic oil diets (SBP r = 0.39 p=0.004; DBP r=0.45, p=0.0006). Moreover, the decrease in central obesity was associated with a reduction in TG on the CanolaOleic oil diet (r = 0.42, p=0.0017). Conclusion: In individuals with and at risk for MetS, consumption of Canola and CanolaOleic oil diets high in MUFA decreased android obesity with an accompanying improvement in MetS risk factors. Our findings demonstrate that oils high in oleic acid may be of benefit 15

29 for the prevention of MetS. 3.2 Introduction Recent estimates indicate that 2.1 billion individuals (27.5% of the world population) are overweight or obese (37). In the U.S., 69% of adults are either overweight or obese, with approximately 35% classified as obese (38). Overweight and obesity increase risk of many health conditions including cancer, cardiovascular diseases, dyslipidemia and type 2 diabetes, as well as other diseases and conditions (39-41). Excess intra-abdominal adipose tissue is related to a clustering of cardiometabolic risk factors known as metabolic syndrome (MetS). Abdominal obesity is a key criterion MetS along with glucose intolerance, dyslipidaemia, and hypertension (42). Also accompanied by inflammation, MetS increases the risk of cardiovascular disease (CVD) and type 2 diabetes mellitus (43). During the past decade, the prevalence of MetS has increased substantially in the US; almost 40% of adults in the US have MetS based on the International Diabetes Federation definition (IDF) (8). For the treatment of MetS, weight loss is the primary therapy (44). The recent AHA/ACC/TOS Guideline for the Management of Overweight and Obesity in Adults suggest a variety of dietary approaches with varying macronutrient profiles as effective for weight loss in overweight and obese adults as long as energy intake is reduced (3). The DASH (Dietary Approaches to Stop Hypertension) dietary pattern is recommended for CVD risk reduction with the option for variations in macronutrient profiles (45). The DASH dietary pattern is low in saturated fatty acids, and one option is high in unsaturated fatty acids. Evidence is emerging that demonstrates beneficial effects of dietary monounsaturated fatty acids (MUFA) in 16

30 regulating body weight and cardiometabolic risk factors (32). The PREDIMED study has shown that long-term consumption of a high MUFA diet provided by extra virgin olive oil and mixed nuts reduced central obesity (46). In addition, studies have shown that consumption of MUFA-enriched diets (21-23% of energy) promote favorable changes in abdominal fat distribution and reduced central obesity (17, 18). In the OmniHeart study, a diet with 21% of energy from MUFA lowered blood pressure and coronary heart disease risk compared with the higher carbohydrate diet (32). Others have shown that high MUFA (20-23% of energy) diets improved the lipid/lipoprotein profile (30, 47, 48), as well as insulin sensitivity and/or glycemic control (30, 49-51). Our understanding of the role that MUFA play in cardiometabolic disease risk reduction is in the early stages, and further research is needed to clarify the specific effects that MUFA have on visceral adiposity which is causally linked to other MetS criteria. With the development and entry into the marketplace of new high-mufa (principally as oleic acid) vegetable oils, it is likely that MUFA intake will increase (52). Consequently, studies are needed to better understand the health effects of MUFA. The objective of the present work was therefore to evaluate the effects of five vegetable oil blends varying in MUFA and PUFA on body composition changes and cardiometabolic risk factors in individuals with or at risk for MetS. Our hypothesis was that high MUFA diets would beneficially affect visceral adipose tissue in individuals with MetS with a corresponding reduction in cardiometabolic risk factors. 17

31 3.3 Methods Participant Characteristics The study was a multi-center, double blind, randomized, cross-over controlled feeding trial. Details about the study methodology have been published previously (53). In brief, participants were recruited at three sites (University of Manitoba, and Laval University, Canada as well as the Pennsylvania State University, USA). Participants were 20 to 65 years of age, had a BMI between 22 to 40 kg/m 2, and were at risk for MetS. Participants had at least one MetS criterion in addition to increased waist circumference (male 94cm, female 80cm) based on the IDF definition for MetS. These included elevated blood glucose ( 5.5mmol/L), decreased HDL-C (male 1.0mmol/L, female 1.3mmol/L), increased triglycerides ( 1.7mmol/L) and elevated blood pressure (SBP 130mmHg or DBP 85mmHg). Participants were recruited via flyers, local newspapers, radio advertisements, and campus mail. The study was approved by the Ethics Committee of University of Manitoba, Laval University and The Pennsylvania State University and carried out in accordance with the Helsinki Declaration. The study design implemented is presented in Figure 3-1. The recruitment flow chart is presented in Figure Experimental Diets Participants were fed an isocaloric diet and requested to follow their routine physical activity practices to maintain body weight. The energy level fed to each 18

32 participant was held constant for all experimental diets. Weight was measured daily (Monday through Friday) when meals were picked up at each diet center. Each treatment period lasted 4 wk and was separated by a 4-wk washout interval, although 10 participants underwent a shortened washout period of 2 to 4 wk at the Pennsylvania State University. The macronutrient profile of the study diets was based on a typical American diet with 50% of the energy from carbohydrate, 35% of energy from fat (18% from treatment oils) and 15% of energy from protein. Five treatment oils were studied: Canola oil (conventional canola oil), CanolaOleic (high-oleic acid canola oil), CanolaDHA (high-oleic acid canola oil with DHA), Corn/Saff (Corn/Safflower oil), and Flax/Saff (Flax/Safflower oil) and incorporated into smoothies that participants consumed twice daily. The fatty acid profiles of treatment oils are presented in Table 1. The energy intake and nutrient profile of the experimental diets are presented in Table 2. One hundred and thirty participants completed the study. A subgroup of participants (n=28) from University of Manitoba site did not receive a metabolically controlled feeding regimen; they prepared their own meals in a communal kitchen under supervision, and consumed the smoothies with the treatment oils that were pre-made and delivered from the University of Manitoba. These participants were excluded from the analysis due to the lack of tight diet control (53). One participant who lost more than 10% of body weight due to illness unrelated to the study was also excluded from the analysis Dual-Energy X-Ray Absorptiometry (DXA) measurements Body composition was assessed pre-intervention on days 1 and 29 by DXA according to the manufacturer s recommendations (Lunar Prodigy Advance, GE 19

33 healthcare, Madison, WI, USA; QDR-4500W; Hologic Corp,Waltham, MA ). Total and regional body composition were determined with Prodigy Encore 2005 software (version ) and APEX System software (version 4.0) using their default configurations. Criteria used to identify the anatomical region of interest were identical across all sites. The android region of interest (ROI) represented the abdominal area. The android ROI started at the pelvic cut line with a height that was 20% of distance from the pelvic cut line to the neck cut line, with the lateral boundaries at the arm cut lines. The gynoid ROI was defined as the area between the top of the femur and mid-thigh with a height two times that of the height of the android region with the lateral boundaries at the leg cut lines. Baseline body composition was available for a subset of study population (n=54). All sites performed DXA scans at the end of each diet period (n=101) Plasma Fatty Acids Plasma fatty acids were extracted using the Folch method with chloroformmethanol (2:1, v/v) containing 0.01% butylated hydroxytoluene (Sigma-Aldrich, Oakville, ON, Canada), followed by methylation with methanolic HCl. Fatty acid methyl esters were then analyzed using an Agilent 6890N gas chromatograph equipped with a flame ionization detector (Agilent Technologies, Mississauga, ON, Canada). During the extraction and methylation, heptadecanoic acid (C17:0) was added as an internal standard (Sigma-Aldrich, Oakville, ON, Canada). Known fatty acid standards (NuChek Prep, Inc. Elysian, MN, USA) were used to identify the individual fatty acids. The amount of each fatty acid was then calculated according to the corresponding peak area relative to the total area of individual fatty acids and presented as the percentage of total fatty acids (53). 20

34 3.4 Statistics Variables were tested for normality and reported as the mean ± standard error. Statistical analyses were performed using the mixed model procedure in the Statistical Analysis Systems statistical software package version 9.2 (SAS Institute, Cary, NC, US). Models included the following factors as fixed effects: diet, period, age, gender, diet by period, diet by gender interaction with participant ID, and center as random effects. There was no indication of a diet carryover effect. Significant diet and diet by gender effects (two-sided p 0.05) were investigated with Tukey post-hoc tests. Differences in baseline characteristics between genders and between subsets and the total study population were determined by ANOVA. Changes from baseline were calculated by subtracting the preintervention measurements from the post-intervention measurements. Bivariate correlations were determined using the Pearson correlation coefficient. Endpoint measurements were compared unless otherwise stated. Regression modeling was performed using SAS regression analysis with the stepwise selection option. Multivariable regression models were used to determine plasma fatty acid concentrations as a predictor of abdominal adipose tissue changes in response to the three high oleic diets (Canola, CanolaOleic, CanolaDHA). Final models were selected on the basis of optimized fit statistics (Smallest Mallow s Cp) and explanatory power (largest R 2 ). 21

35 3.5 Results Participant Characteristics Energy intake was the same across the five treatment diets during each 4-wk diet period (Table 2). Baseline anthropometric and metabolic characteristics of participants are summarized in Table 3. One hundred and one participants (50 males and 51 females) were included in the analyses. Males had a greater body mass (p<0.0001) and waist circumference (p<0.0001) compared with females at baseline. According to self-reported physical activity log, participants maintained a consistent level of physical activity throughout the study (not shown). In addition to an increased waist circumference, 57 participants had only one additional MetS criterion (M=25, F=32), 24 participants had two criteria (M=17, F=7), 19 participants had three criteria (M=7, F=12), and one male participant had four criteria. Thus, all participants had at least one MetS criterion in addition to central obesity, which characterized them as a population with or at risk for MetS. Body composition at baseline is presented in Table 4. A subset of participants (n=54) who had a baseline body composition assessment were compared with the total study population (n=101) in baseline anthropometric characteristics and cardiometabolic markers. There were no significant differences in body weight, BMI, cholesterol, HDL-C, triglycerides, CRP, SBP and DBP between these two groups at baseline. Also, at the end of each diet period, the changes in cardiometabolic risk factors from baseline did not differ between the subset participants and all study participants. There was no significant difference in android fat mass across all five diets between the subset participants and the total study population (Data not shown). 22

36 Baseline correlations between adipose tissue mass and cardiometabolic risk factors are presented in Table 5. Android fat mass was positively correlated with BMI (r=0.86, p<0.0001), SBP (r=0.32, p=0.0225) and DBP (r=0.32, p=0.0186), CRP (r=0.28, p=0.0425) and TG levels (r=0.27, p =0.0447) Diet-specific Effects on the Plasma Fatty Acid Profile Plasma fatty acid profiles of COMIT I participants (n=130) have been reported previously (53) and reflect the fatty acid content of the five vegetable oils consumed. The fatty acid data presented herein (n=101) confirm good dietary adherence (Supplemental Table 1). Between-diet comparisons revealed that total MUFA content was the highest in participants on the CanolaOleic oil diet, followed by the Canola oil diet versus the other diets (p<0.0001, for both). The CanolaDHA oil diet also increased plasma MUFA (13.16% ± 0.022) compared with the Corn/Saff (11.65% ± 0.022, p<0.0001) and Flax/Saff oil diets (11.96%±0.022, p<0.001). The Corn/Saff oil diet resulted in the highest total n-6 PUFA concentration (38.73%±0.25, p<0.0001), followed by the Flax/Saff oil diet (34.66%±0.25, p=0.0043). In both of these diets, linoleic acid was higher compared with the other three MUFAenriched diets. The Flax/Saff oil diet resulted in the highest ALA content (1.68±0.037, p<0.0001). Between-diet comparisons showed that consumption of the CanolaDHA oil diet was associated with the greatest total n-3 PUFA plasma concentration (9.61%±0.11), mainly contributed by EPA (1.62%±0.056) and DHA (7.09%±0.08). Plasma DHA concentrations were similar for all other diets. 23

37 3.5.3 Diet-specific Effects on Body Weight and Body Composition Body composition results are presented in Table 6. At the end of each diet period, total fat mass, total lean mass, trunk lean mass, android lean mass, gynoid fat mass and gynoid lean mass remained unchanged for all treatments. Minor body weight fluctuations were observed in the study. Participants had lower body weights at the end of the Canola (p=0.0072) and CanolaOleic oil diets (p=0.016) compared to the Flax/Saff oil diet. There was a strong trend for the Canola oil diet to decrease trunk fat mass compared with the Flax/Saff oil diet (p=0.052). Gender differences were noted for trunk fat mass in response to the different diets (p=0.047 for interaction). In males, the CanolaOleic oil diet decreased trunk fat mass compared with the Flax/Saff oil diet (p=0.036). In contrast, the Canola oil diet decreased trunk fat mass by 0.48kg compared with the Corn/Saff oil diet (p=0.043) in females. A main diet effect on android fat mass was also observed (p = ). In all participants, android fat mass was reduced by 0.06kg (66.09g) on the CanolaOleic oil diet (p=0.026), and 0.05kg (54.59g) the Canola oil diet (p=0.025) compared with the Flax/Saff oil diet. The decrease in android fat mass was affected by gender (p=0.033 for interaction). Specifically, in males, the CanolaOleic oil diet decreased android fat mass by 0.13kg (p=0.0027) as well as the android-to-gynoid fat mass ratio (p=0.0067) compared with the Flax/Saff oil diet. In contrast, there were no differences in females across the five treatment diets. 24

38 3.5.4 Diet-specific Effects on Changes in Body Weight and Body Composition from Baseline Body composition changes from baseline are summarized in Figure 3-3. Body weight decreased significantly from baseline in participants on all diets (Canola, -0.91kg, p = ; CanolaOleic, -1.18kg, p=0.0001; CanolaDHA -0.96kg, p = ; Corn/Saff,-0.79kg, p =0.0075) except the Flax/Saff oil diet (-0.33kg, p=0.0687). There was a tendency for the CanolaOleic oil diet to decrease body weight from baseline to a greater extent compared with the Flax/Saff oil diet (p=0.074). Total fat mass remained unchanged between these two diets, however, a strong tendency was observed (p=0.0582) for the CanolaOleic oil diet to reduce total fat mass from baseline. The Flax/Saff oil diet preserved total lean mass from baseline compared with the other diets (Supplemental Figure 3-4A). Android fat mass was decreased from baseline on the Canola oil (p=0.042) and CanolaOleic oil diets (p=0.007); the reduction in response to the CanolaOleic oil diet was greater than that for the Flax/Saff oil diet (p=0.0298) Figure 3-4B. No between diet differences were observed among the Canola, CanolaDHA and Corn/Saff oil diets for android fat mass. A gender difference was observed for android fat mass in response to the experimental diets (p=0.0203). Male participants experienced a significant android fat mass loss (p=0.017) after the CanolaOleic oil diet compared with the Flax/Saff oil diet. At the end of each diet, android lean mass, and trunk lean mass remained unchanged for all treatments (Data not shown). All diets decreased gynoid lean mass from baseline except the Corn/Saff oil diet (Supplemental Figure 3-4B). 25

39 3.5.4 Correlation between Android Fat Mass Changes and Cardiometabolic Risk Factors in Response to Diet Correlations between changes in android fat mass and cardiometabolic risk factors in response to the different diets are presented in Table 7. Changes in android fat mass from baseline after the Canola and CanolaOleic oil diets were positively associated with changes in SBP (Canola, r=0.26, p=0.0624; CanolaOleic, r=0.39, p= and DBP (Canola, r=0.38, p=0.0049; CanolaOleic, r=0.45, p=0.0006). The changes in android fat mass were positively correlated with triglyceride levels after consumption of the CanolaOleic oil (r=0.42, p=0.0017) and the Flax/Saff oil (r=0.41, p=0.002) diets Sub-analysis Between Genders and Plasma Fatty Acid Levels Results from regression modeling on the relationship between plasma fatty acid concentrations and android fat mass changes in a subgroup of the study population in response to the Canola, CanolaOleic and CanolaDHA oil diets are presented in Supplemental Table 2. Android fat mass changes were partially determined by plasma oleic acid content (R 2 = 19.23%, p = ) and nervonic acid concentration (R 2 = 16.61%, p=0.038) in males in response to the CanolaOleic oil diet. Similarly, nervonic acid concentration was an independent predictor in abdominal fat mass changes in females in response to the CanolaOleic diet (R 2 =19%, p=0.0099). 26

40 3.6 Discussion Our findings demonstrated that CanolaOleic oil and Canola oil rich diets reduced android fat mass compared to a n-6 PUFA-enriched Flax/Saff oil diet when each was provided at identical energy levels for 28d. The android fat mass changes were accompanied by a reduction in the android to gynoid fat mass ratio after the CanolaOleic oil diet in males. The reduction was due to a decrease in central body fat mass, rather than a redistribution of adipose tissue to the lower body as indicated by an absence of change in the gynoid fat mass. Importantly, the CanolaOleic oil diet provided the most MUFA (19.3% of energy) compared with the Canola and CanolaDHA oil diets that also were high in MUFA (17.6% and 17.8% of energy, respectively). In the two diets that decreased android fat mass, plasma oleic acid concentration was highest (Canola, 14.9%; CanolaOleic, 15.6%) compared with the other treatment oil diets, including the CanolaDHA oil diet (13.7%). These findings add to the evidence base that dietary MUFA beneficially affects central adiposity in a calorie-controlled setting. In support of our finding, Paniagua et al. (17) reported that a MUFA-rich diet did not affect central adipose tissue redistribution whereas the carbohydrate-rich diet promoted adverse adipose tissue redistribution towards the android depot concomitant with a decrease in peripheral adipose tissue. Similarly, Gillingham et al. (18) reported a tendency for a reduction (p = 0.055) in the android to gynoid fat mass ratio after consumption of a high oleic canola oil diet that provided 21% MUFA from canola oil for 28 days in hypercholesterolemic individuals. In the present study, the CanolaOleic oil diet reduced both body weight (1.18kg) and android fat mass (0.11kg) in just four weeks. Whether longer-term consumption of MUFA elicits greater weight and body composition changes remains to be evaluated. 27

41 The PREDIMED study has shown a decrease in waist circumference, which is a surrogate marker for abdominal adiposity, and demonstrated cardiovascular benefits in response to long term consumption of Mediterranean diets supplemented with extra virgin olive oil or mixed nuts (46, 54, 55). The mechanisms for the reduction in body weight and android fat mass may include a greater oxidation rate and/or increased energy expenditure in response to the consumption of a high MUFA diet. Previous studies have shown that diets enriched in MUFA affect fat balance, body mass and possibly energy expenditure (15, 56, 57). Paniagua et al. reported that compared to a carbohydrate-rich diet, a MUFA-rich diet resulted in greater fat oxidation rates and a decrease in the abdomen-to-leg adipose tissue ratio in insulin resistant participants (17). In addition, Schmidt et al. (24) reported that the fractional oxidation rate of oleic acid was significantly greater (21%) than for palmitic acid in healthy men (n = 10) after consuming meals that provided 16.5% energy from oleate and 16.3% from palmitate during a 8 hour postprandial period. Kien et al. (56) demonstrated that consumption (for 28 days) of a high oleic acid diet (1.7% palmitic acid and 31.4% oleic acid) increased the fatty acid oxidation rate in women compared to the control diet (8.4% palmitic acid and 13.1% oleic acid). In the same study, a high oleic acid diet increased daily energy expenditure compared to the high palmitic acid diet (16.8% palmitic acid, 16.4% oleic acid) in men (56). Collectively, the results from other studies are suggestive of increased oxidation and energy expenditure mechanisms that may explain the decrease in body weight and android fat mass in response to the two high oleic acid diets in the present study. The increase we observed in plasma MUFA concentration on the CanolaOleic oil and Canola oil diets could have increased the fatty 28

42 acid oxidation rate leading to an increase in energy expenditure. Related to this, MUFA has been shown to activate PPAR-delta and thereby increase fatty acid oxidative capacity (58, 59), a finding that provides a plausible molecular mechanism for the body weight and android fat mass decreases we report herein for the highest MUFA diets. Moreover, a derivative of oleic acid in small intestine, oleoylethanolamide (OEA), has anorectic properties, thereby decreasing appetite and food intake (60). This is not applicable to the present study, however, since all food was provided to our participants. OEA also has been shown to induce lipolysis through the activation of PPAR-α, which provides another possible mechanism of action for the favorable body composition changes in participants on the high MUFA diets (61-64). Also of interest are two in vitro rodent adipose tissue studies that have shown that MUFA stimulate lipolysis (65, 66). Further research is needed to better understand all of the underlying mechanisms that account for the effects of MUFA on body composition. An intriguing observation is the negative relationship between plasma nervonic acid concentration and android fat mass reduction in response to the CanolaOleic oil diet. Nervonic acid is derived from oleic acid by chain elongation (67). Evidence has shown that serum nervonic acid concentration is negatively associated with MetS risk (68). One small cross-sectional study from Japan reported an inverse association between plasma nervonic acid and circulating leptin levels (69). The negative correlation between plasma oleic acid concentration and android fat mass reduction in males but not females suggests a gender-specific response to a high MUFA diet. However, the mechanism by which nervonic acid is associated with adipose tissue reduction in females in the present study is 29

43 unclear. Additional research is needed to clarify the role that nervonic acid may have on adipose tissue mass. Numerous studies have shown that android tissue mass is positively correlated with blood pressure, triglycerides and CRP levels (2, 70, 71). In the present study, we demonstrated that the reduction in android fat mass was positively correlated with decreases in cardiometabolic risk factors including triglycerides, SBP and DBP after the Canola, CanolaOleic, CanolaDHA, and Flax/Saff oil diets, but not the Corn/Saff oil diet. Previous evidence has shown that dietary MUFA decrease SBP (32). A recent crosssectional study of 4680 men and women in China, Japan, UK and the US reported that MUFA intake (ranging from 8.1% kcal to 12.2% kcal), especially oleic acid from vegetable oil sources, may contribute to the prevention of high blood pressure (72). Research has shown that weight loss and a reduction in android adiposity decrease MetS risk (71, 73). The positive correlation between the reduction in android fat mass and the decrease in cardiometabolic risk factors demonstrates unique effects of dietary MUFA on MetS criteria that could be due to a decrease in visceral adipose tissue. Fatty acids have depot-specific effects on lipid accumulation (74) in that MUFA are taken up more readily by the peripheral tissues compared to SFA, and, consequently, represent the principal fatty acid in the lipid component of adipose tissue (75). MUFA preferentially accumulate in subcutaneous adipose tissue in contrast to SFA that preferentially accumulate in visceral adipose tissue (74-76). Consumption of a high oleic acid diet may contribute to preferential fat deposition in subcutaneous adipose tissue and thereby decrease visceral adiposity and accompanying adverse metabolic effects. Thus, the well-established 30

44 benefits of MUFA in decreasing MetS risk (71, 73) may be due at least partly to this redistribution of fat deposition between subcutaneous and visceral adipose tissues. A strength of the present study is the controlled diet design, including the delivery of the test oils, all of which were incorporated into smoothies for easy and accurate administration of the specified dose of oil. In addition, the cross-over study design minimized the influence of genetic polymorphisms that contribute to variations in diet responsiveness and consequently inter-individual differences in results among study participants (77). Potential limitations of the study include lack of baseline body composition data for all participants resulting in less statistical power for assessing body composition changes from baseline. Another important limitation is that we conducted a controlled isocaloric feeding study, which may have attenuated the impact of a selfselected high MUFA diet on energy intake (and expenditure). 3.7 Conclusion In summary, short-term consumption of diets high in MUFA provided by Canola oil and CanolaOleic oil was associated with a reduced body weight (-1.18kg) and android adipose tissue mass (-0.11kg) in participants with or at risk for MetS. These changes were associated with favorable shifts in cardiometabolic risk factors. Importantly, our findings provide evidence for a beneficial effect of dietary MUFA in lowering cardiometabolic risk that we suggest is mediated by a decrease in visceral adipose tissue mass. 31

45 Figure 3-1. Study Design for COMIT Canola CanolaOleic CanolaDHA Diet Period 1 Diet Period 2 Diet Period 3 Diet Period 4 Diet Period 5 Corn/Saff Flax/Saff Blood Draw, Anthropometric measurement, DXA, MRI (n=14) 32

46 Figure 3-2. Recruitment Flowchart Primary Screening (n=1112) Clinical Screening (n=704) Excluded (n=534) Not meeting inclusion criteria (n=369) Declined to participate (n=165) Randomized (n=170) Dropouts (n=40) Illness unrelated to study diet (n=8) Diet issues (n=8) Participants burden (n =6) Family/job/relocation (n=9) Violation of study protocol (n =4) Pregnancy (n =1) Loss of interest (n = 2) Unknown reason (n =2) Completed (n=130) RCFFN (n=69) INAF (n=58) PSU (n=43) RCFFN (n=54) INAF (n=46) PSU (n=30) Excluded from analyses Lack of metabolically diet control (n=27) Loss >5% body weight (n=2) Analyzed (n=101) RCFFN (n=26) INAF (n=46) PSU (n=29) 33

47 Table 3-1. Fatty acid profiles of the five treatment oils Canola CanolaOleic CanolaDHA Corn/Saff Flax/Saff Fatty Acids Percentage SFA MUFA PUFA Omega-3 ALA < DHA Omega-6 LA

48 Table 3-2. Energy and nutrient profiles of the experimental diets Canola CanolaOleic CanolaDHA Corn/Saff Flax/Saff Energy intake (kcal)* ± ± ± ± ±60.4 Nutrient profiles Protein 15.6% 15.6% 15.6% 15.6% 15.6% Carbohydrate 50.7% 50.7% 50.7% 50.7% 50.7% Fat 35.5% 35.5% 35.5% 35.5% 35.5% SFA 6.6% 6.5% 6.9% 6.7% 6.8% MUFA 17.6% 19.3% 17.8% 9.5% 9.6% Oleic 15.7% 18.0% 16.5% 8.3% 8.4% PUFA 9.1% 6.9% 8.0% 16.3% 16.3% ALA 2.0% <1.0% <1.0% <1.0% 6.0% Omega-3 DHA 1.1% Omega-6 LA 6.5% 5.6% 5.2% 15.4% 9.7% * Values are expressed as means ± SEM 35

49 Table 3-3. Metabolic characteristics of participants at screening Characteristic Anthropometric measurements All participants (n=101) Males (n=50) Females (n=51) Age 49.5± ± ±1.5 Body mass (kg) 85.8± ±1.9 * 75.9±2.1 * Height (m) 1.7± ± ±0.01 Body mass index (kg/m2) 29.4± ± ±0.6 Waist circumference 101.9± ±1.2 * 96.8±1.4 * Metabolic syndrome risk factors Glucose (mmol/l) 5.24± ± ±0.20 HDL-cholesterol (mmol/l) 1.25± ± ±0.05 Triglycerides (mmol/l) 1.84± ± ±0.12 Blood pressure SBP/DPB(mmHg) 122/78 123/78 118/78 Number of Metabolic syndrome risk factors per participant 1 factor factors factors factors 1 1 Values are expressed as means ± SEM * Values were significantly different between genders p<

50 Table 3-4. Body composition of participants at baseline All participants (n=54) Males (n=20) Females (n=34) Body fat mass (kg) 32.95± ± ±1.48 Body lean mass (kg) 51.32± ±2.02 * 43.33±0.89 Body fat mass (%) 38.93± ± ±1.22 Body lean mass (%) 60.11± ±1.35 * 56.55±1.24 Trunk fat mass (kg) 17.18± ±1.14 * 16.40±0.82 Trunk lean mass (kg) 24.93± ±1.10 * 21.15±0.51 Android fat mass (kg) 3.13± ±0.227 * 2.93±0.16 Android lean mass (kg) 3.70± ±0.141 * 3.13±0.08 Gynoid fat mass (kg) 5.56± ± ±0.28 Gynoid lean mass (kg) 7.89± ±0.318 * 6.67±0.16 Values are expressed as means ± SEM * Values were significantly different between gender p<

51 Table 3-5. Correlation between adipose tissue mass and cardiovascular risk factors at baseline (n=54) Total fat mass Android fat mass Gynoid fat mass Android/ Gynoid fat mass r p-value r p-value r p-value r p-value BMI 0.85 < < < CHOL NS NS NS NS HDL-C 0.19 NS 0.01 NS TG NS NS CRP NS 0.13 NS SBP 0.16 NS NS DBP 0.11 NS NS NS: non-significant 38

52 Table 3-6. Endpoint body weight and body composition after each of the experimental diets (n=101; males n=50, females n=51) Body composition measurements Canola CanolaOleic CanolaDHA Corn/Saff Flax/Saff p-value (kg) Body mass All 86.60±1.53 a 86.75±1.53 a 86.89±1.53 ab 86.96±1.53 ab 87.28±1.56 b Male 95.32±1.19 ab 94.96±1.92 a 95.42±1.90 ab 95.47±1.93 ab 95.9±1.92 b ǂ Female 76.81± ± ± ± ±1.59 NS All 31.21± ± ± ± ± Total fat mass Male 30.71± ± ± ± ±1.15 NS Female 31.72± ± ± ± ±1.16 NS All 51.32± ± ± ± ± Total lean mass Male 60.70± ± ± ± ±1.17 NS Female 42.13± ± ± ± ±0.69 NS All 16.73± ± ± ± ± Trunk fat mass Male 17.90±0.67 ab 17.63±0.65 a 17.87±0.68 ab 17.88±0.68 ab 18.19±0.69 b ǂ Female 15.59±0.66 a 15.78±0.67 ab 15.88±0.68 ab 16.07±0.69 b 15.79±0.66 ab ǂ All 24.17± ± ± ± ± Trunk lean mass Male 28.21± ± ± ± ±0.70 NS Female 20.20± ± ± ± ±0.44 NS All 3.11±0.096 a 3.09±0.094 a 3.15±0.096 ab 3.14±0.094 ab 3.16±0.097 b ǂ Android fat mass Male 3.38±0.14 ab 3.32±0.14 a 3.38±0.13 ab 3.37±0.13 ab 3.45±0.14 b ǂ Female 2.85± ± ± ± ±0.12 NS All 3.62± ± ± ± ± Android lean mass Male 4.27± ± ± ± ±0.10 NS Female 2.98± ± ± ± ±0.06 NS All 5.27± ± ± ± ± Gynoid fat mass Male 4.86± ± ± ± ±0.24 NS Female 5.67± ± ± ± ±0.21 NS 39

53 All 7.68± ± ± ± ± Gynoid lean mass Male 9.02± ± ± ± ±0.19 NS Female 6.36± ± ± ± ±0.14 NS All 0.61± ± ± ± ± A/G Male 0.72±0.02 ab 0.71±0.02 a 0.72±0.02 ab 0.72±0.03 ab 0.73±0.03 b ǂ Female 0.51± ± ± ± ±0.02 NS Values are expressed as means ± SEM. Values in a row with different superscript letters indicate significant differences between diets p<0.05 (Mixed model post hoc analysis of significant diet by gender effect with tukey adjustment) 1 Canola vs. Flax/Saff (p=0.0518) NS: non-significant Android-to-gynoid fat mass ratio 40

54 Kg kg Figure 3-3. Body weight android fat mass and trunk fat mass changes in response to five experimental diets (n=54; males n=20, females n=34) A. 0 Body Weight All * * * * Male Female -2.5 Canola CanolaOleic CanolaDHA Corn/Saff Flax/Saff B Android Fat Mass * a b a b * a * a a b a b a b a b b b All Male Female -0.4 Canola CanolaOleic CanolaDHA Corn/Saff Flax/Saff 41

55 Kg C. 0.5 Trunk Fat Mass 0.0 All * * Male Female -1.5 Canola CanolaOleic CanolaDHA Corn/Saff Flax/Saff Values with different superscript letters indicate significant differences between diets p<0.05 (Mixed model with tukey post-hoc test). * Values were significantly different from baseline value. 42

56 Table 3-7. Correlation between android fat mass changes and changes in cardiovascular risk factors (n=54) Canola CanolaOleic CanolaDHA Corn/Saff Flax/Saff Changes from Android fat mass baseline r p-value r p-value r p-value r p-value r p-value CHOL 0.10 NS 0.15 NS NS 0.08 NS NS HDL NS NS 0.11 NS 0.12 NS NS TG 0.07 NS NS 0.26 NS CRP NS NS 0.11 NS 0.11 NS 0.19 NS SBP NS DBP NS

57 3.8 Supplemental Material Table Plasma fatty acid profiles in response to the experimental diets Total Fatty Acids (n=101) Canola CanolaOleic CanolaDHA Corn/Saff Flax/Saff Percentage of all fatty acids ΣSFA 42.03±0.16 a 41.84±0.16 a 43.04±0.16 b 42.81±0.16 b 42.86±0.16 b ΣMUFA 17.85±0.24 a 18.52±0.25 b 16.37±0.25 c 14.22±0.25 d 14.47±0.24 d C14:1n5 0.13± ± ± ± ±0.023 C16:1n7 1.06±0.050 ac 1.08±0.051 c 0.89±0.051 b 0.94±0.051 b 0.99±0.050 ab C18:1n ±0.022 a 15.62±0.022 b 13.69±0.022 c 11.65±0.022 d 11.96±0.022 d C20:1n9 0.30±0.022 ab 0.34±0.023 a 0.27±0.023 abc 0.23±0.023 bc 0.19±0.022 c C24:1n9 1.41± ± ± ± ±0.046 Σn-6PUFA ±0.25 a ±0.25 a 30.97±0.25 b 38.73±0.25 c 35.91±0.25 d C18:2n ±0.24 a 21.54±0.24 a 19.05±0.24 b 25.84±0.24 c ±0.24 c C18:3n6 0.17±0.013 a 0.18 ±0.013 a ±0.013 b 0.18 ±0.013 a 0.12 ±0.013 b C20:2n6 0.31±0.04 ab 0.33±0.038 ab 0.19±0.038 b 0.36±0.038 a 0.32±0.038 ab C20:3n ±0.084 a 2.58±0.085 a 1.75±0.085 b 2.29±0.085 a 1.88 ±0.084 b C20:4n6 9.49±0.18 a 9.75±0.18 a 9.67±0.18 a 9.69±0.18 a 8.12±0.18 b C22:4n6 0.25±0.033 ab 0.034±0.034 ab 0.22±0.033 b 0.37±0.034 a 0.17±0.033 b Σn-3PUFA 5.55±0.11 a 4.97±0.11 b 9.61±0.11 c 4.23±0.11 d 6.75±0.11 e C18:3n3 0.83±0.037 a 0.68±0.037 b 0.61±0.037 bc 0.53±0.037 c 1.68±0.037 d C20:5n3 1.13±0.054 a 0.88±0.056 b 1.62±0.056 c 0.51±0.055 d 1.51±0.055 c C22:5n3 0.77±0.031 a 0.69±0.031 ac 0.30±0.031 b 0.60±0.031 c 0.96±0.031 d C22:6n3 2.83±0.080 a 2.72±0.08 a 7.09±0.08 b 2.60±0.08 a 2.60±0.08 a Endpoint values are expressed as means ± SEM. Different superscript letters indicate significant differences between diets p<

58 Kg Kg Figure 3-4. Total lean mass and gynoid lean mass changes in response to five experimental diets (n=54; males n=20, females n=34) A. 0.5 Total Lean Mass * * * * All Male Female -1.5 * Canola CanolaOleic CanolaDHA Corn/Saff Flax/Saff B. 0.5 Gynoid Lean Mass * * * * * * * * All Male Female -1.0 Canola CanolaOleic CanolaDHA Corn/Saff Flax/Saff * Values were significantly different from baseline value. 45

59 Table Plasma fatty acids as predictors of android fat mass changes in response to Canola, CanolaOleic, CanolaDHA oil diets Canola Predictor Coefficient ± SEM p-value Model R 2 Female Nervonic acid (C24:1n9) ± CanolaOleic Predictor Coefficient ± SEM p-value Model R 2 Female Nervonic acid (C24:1n9) ± Palmitic acid (C16:1n7) ± Male Oleic acid (C18:1n9) ± Nervonic acid (C24:1n9) ± CanolaDHA Predictor Coefficient ± SEM p-value Model R 2 Male Oleic acid (C18:1n9) ± Values are expressed as coefficient ± SEM. 46

60 Chapter 4 LITERATURE REVIEW 4.1 Introduction In 1951, Barr et al. first reported reduced plasma levels of HDL-C in patients with CHD (78, 79). Later, numerous prospective epidemiologic studies and intervention studies demonstrated a strong inverse relationship between levels of HDL-C and CHD risk (80). In 1977, Miller et al. initially demonstrated the importance of low HDL-C as risk factor for CHD (81), and this finding has been repeatedly confirmed. In 1989, a meta-analysis of four prospective studies (the Framingham Heart Study, the Multiple Risk Factor Intervention Trial, the Lipid Research Clinics Prevalence Mortality Follow-up Study and the Coronary Primary Prevention Trial) reported that a 1 mg/dl (0.026 mmol/l) increase in HDL-C was associated with a significant decrease in CHD risk for men (2%) and women (3%) (80). These important findings have led to a large number of investigations over the last few decades to evaluate the role of HDL-C in modulating CVD risk. In 2004, the National Cholesterol Education Program Adult Treatment Panel III Guidelines defined an HDL-C of <40 mg/dl as an independent risk factor for cardiovascular disease (CVD) (82). Dietary interventions have been shown to affect HDL-C concentrations. It is well documented that replacing CHO with MUFA or PUFA increases HDL-C by about 3.4mg/dl and 2.8mg/dl, respectively (22, 83). The Mediterranean dietary pattern which emphasizes olive oil, nuts, fruits, vegetables and whole grains has been shown to have beneficial effects on CVD progression by, in part, increasing HDL-C levels (84, 85). Some foods contain 47

61 bioactive compounds that can also improve the lipid/lipoprotein profile in different population by elevating HDL-C levels (86-92). In addition to HDL-C levels, recent research suggests that more attention should be paid to HDL function. HDL has been proposed to have several anti-atherosclerotic properties, including the ability to mediate macrophage cholesterol efflux, increase antioxidant capacity and exert antiinflammatory effects (93). HDL particles are critical acceptors of cholesterol from lipid loaded macrophages. The concentration of HDL-C provides information about the size of the HDL pool, but does not convey anything about HDL function. Recently, in vitro, ex vivo evidence suggests that some bioactive compounds are effective in increasing the capacity of HDL to mediate cholesterol efflux from macrophages by increasing cholesterol transporter protein expression in macrophage plasma membranes. This review will examine the effects of diet on HDL-C levels and HDL function and also explore the mechanisms by which bioactive compounds improve cholesterol efflux capacity. 4.2 The HDL-C Hypothesis Controversy In the 1980s, the Framingham study provided the first insight into the relationship between LDL-C levels, HDL-C levels and CVD risk. This finding established the foundation for identifying elevated serum cholesterol as a risk factor for CVD (94). Later, with the advent of statin therapy, researchers found that reduced LDL-C levels significantly decreased CHD risk by 25-30% (95, 96). These findings have fueled interest in better understanding of what can be done to further reduce CHD risk when LDL-C levels are normal, i.e., can CHD risk be further reduced by raising HDL-C levels? This is referred to as the HDL hypothesis. 48

62 Data from the participants in the Framingham study have shown a significant increase in CHD risk in individuals with a normal LDL-C but reduced HDL. Thus, HDL-C was established as an independent risk factor for CHD (94). During the past decade, therapeutic targets that increase the plasma HDL-C level have been investigated. Following the discovery of individuals with a genetic deficiency of cholesteryl ester transfer protein (CETP) who had remarkably elevated HDL levels, inhibiting CETP became a popular therapeutic strategy in (97). Recent clinical trials have questioned the validity of the HDL hypothesis. Two trials, the AIM-HIGH study and the dal-outcomes study evaluated the effects of therapeutic elevations of HDL-C with niacin and dalcetrapib (CETP inhibitor drug) in individuals with low LDL-C and were prematurely terminated due to the lack of benefits (98, 99). Despite a 25% increase in HDL-C after 2 years of niacin therapy in the AIM-HIGH study and nearly a 30% HDL-C increase following intervention with the HDL-C-boosting drug, dalcetrapib (dal-outcomes), there was no significant reduction in major cardiovascular outcomes (98, 99). More recently, Merck globally withdrew their HDL-C elevating drug, Tredaptive (niacin/laropiprant) from the market; this was seemingly based on the null outcome of the recently completed HPS2- THRIVE (Heart Protection Study 2-Treatment of HDL to Reduce the Incidence of Vascular Events) study(100). HPS-2 THRIVE was a randomized trial of the long-term clinical effects of raising HDL-C with Tredaptive (combination of niacin and laropiprant). This study assessed the clinical effects of a combination of 2 g niacin plus 40 mg laropiprant compared with placebo in 20,000 patients with pre-existing atherosclerotic vascular disease. All patients in the study also received 40 mg of a cholesterol lowering drug (Tredaptive ). This trial did not reduce major vascular events after a median follow up of 3.9 years. Instead, there was an 49

63 increase in the incidence of adverse events in the Tredaptive group. These findings suggest that focusing on elevating HDL-C levels solely is not sufficient to lower CHD risk. Thus, it is important to focus on HDL function. Growing evidence suggests that HDL functionality, specifically HDL-mediated cholesterol efflux from macrophages during reverse cholesterol transport (RCT) seems to correlate better with atherosclerotic burden than HDL-C levels (101). Recent research has demonstrated beneficial effects of dietary bioactive components on increasing HDL function ( ). The effects of dietary bioactive compounds on HDL functionality as measured by reverse cholesterol transport (RCT) will be discussed. 4.3 HDL and Reverse Cholesterol Transport Coronary heart disease is characterized by the narrowing or blockage of the coronary arteries, usually caused by atherosclerosis. Atherosclerosis is a condition of plaque build-up in the arterial wall. Atherosclerosis also is considered to be an inflammatory response of macrophages and lymphocytes to invading pathogenic lipoproteins in the arterial wall (105). In the atherosclerotic region, macrophages are the primary cell overloaded with cholesterol. Thus, much research on atherosclerosis has focused on assessing macrophage specific cholesterol efflux (105). The cholesterol deposition in macrophages is first initiated with monocyte recruitment to the vascular lesion sites by cell adhesion molecules (106). The adherent monocytes then migrate into the sub-endothelial space where they differentiate further into macrophages (106). During this differentiation, receptors for ox-ldl are up-regulated. The macrophage 50

64 oxldl is then delivered to the lysosome where cholesterol ester (CE) is hydrolyzed to free cholesterol and fatty acids (106). Cholesterol homeostasis is critical for the cell. The reverse cholesterol transport function of HDL is important for cell cholesterol homeostasis which is thought to be a protective mechanism against atherosclerosis. With respect to RCT, HDL facilitates uptake of cholesterol from the macrophage for return to the liver for excretion into the bile and feces Figure 4-1. Table 4-1. summarizes the pathways for macrophage-specific cholesterol efflux. The major cholesterol efflux mechanisms are: 1) free cholesterol (FC) efflux to the HDL particles via passive diffusion through the plasma membrane (107); 2) cholesterol is effluxed to lipid-poor apoa-1 or HDL particles by ABCA1 or ABCG1 transporters (107, 108); and 3) cholesterol efflux occurs via scavenger receptor class B type I receptors (SR-BI) (109). SR-BI transport of cholesterol both out of and into cells depends on the intracellular cholesterol concentration (110, 111). ABCA1 and ABCG1 are both regulated by the nuclear receptor liver X receptor (LXR-α) and LXR-β (112). Cellular cholesterol generates formation of oxysterol (OS) which is the endogenous ligand for LXRs (113). Increasing cellular cholesterol levels leads to formation of oxysterol ligand for LXRs which are responsive for up-regulating the membrane transporter protein, ABCA1 and ABCGA (113). LXRs are currently considered to be the master regulators of macrophage cholesterol efflux (114). Interestingly, recent evidence reveals that the peroxisome proliferator- activated receptors (PPARs) up-regulate LXRs thus promoting macrophage cholesterol efflux (115). More research is focusing on how PPARs and LXRs work as targets for up-regulating cholesterol efflux from macrophages. Figure 4-2. shows the schematic LXR regulatory cholesterol efflux pathway from the macrophage. 51

65 Table 4-1. Pathways for macrophage-specific cholesterol efflux Efflux pathway Efflux method Cholesterol Process Acceptor Aqueous diffusion Passive HDL Process is bi-directional SR--BI Passive HDL ABCG1 Active transport HDL ABCA1 Active transport Pre-β HDL Process is bi-directional High-affinity receptor for HDL Process is unidirectional No high affinity binding Process is unidirectional Pre-β HDL interaction with high-affinity ABCA1 receptor 52

66 Figure 4-1. Reverse cholesterol transport Source (116) Figure 4-2. Illustration of LXR regulatory cholesterol efflux pathway from macrophages 53

67 4.4 Effect of Dietary Bioactive Compounds on HDL-C Levels Dietary manipulations have multiple effects on many CHD risk factors. The effects of dietary bioactive compounds on HDL-C levels and HDL function are described below. When HDL-C is measured clinically, the cholesterol content contained within the HDL particle is quantified (117). Clinical evidence suggests that bioactive compounds from berries, cocoa and soy products increase HDL-C levels (86-92, 118). A study conducted by Erlund et al. reported that consumption of mixed berries for 8 weeks increased HDL-C levels by 5.2% in subjects with CVD risk factors (86). Based on our current knowledge of the health effects of bioactive components in berries, the authors proposed that polyphenols contributed to the improvement in blood lipids. In male subjects with abdominal obesity, consumption of 250 ml/d and 550 ml/d cranberry juice cocktail for 4 weeks was associated with increases in plasma HDL-C of 8.6% and 8.1%, respectively (90). Cocoa, grapes and orange juice (87, 88, 91, 92) consumption also have been reported to increase HDL-C levels. Table 4-2. summarizes randomized controlled trials evaluating the effects of bioactive compounds on HDL-C concentrations. Polyphenols have been the most studied bioactive compound. Intakes ranging from mg to 840 mg increase HDL-C levels. In addition, isoflavones increase HDL-C. Taken together, bioactive compounds such as polyphenols appear to be effective in increasing HDL-C concentrations and, thereby improving cardiovascular health. 54

68 Table 4-2. Effects of bioactive compounds on HDL-C levels in randomized controlled Trials References Erlund et al. (86) Baba et al. (87) Mursu et al. (88) Baum et al. (89) Ruel et al. (90) Kurowska et al. (91) Hansen et al. (92) Design SB, P Subject characteristic Location (M/F)n Length Finland M/n=25 F/n=46 8 wk SB, P Japan M/n=25 12 wk P Finland M/n=12 F/ n=33 3 wk DB, P USA F/n=66 6 mo SB,CO Canada M/n=31 4-wk CO P Canada Denmark M/n=16 F/n=9 M/n=31 F/n=38 4 wk 4 wk SB, single-blind; P, Parallel; DP, double-blind; CO, cross-over Food sources/ bioactive compounds Berry/ polyphenol Cocoa/ polyphenols Chocolate/ polyphenols Soybeans/ isoflavone Cranberry juice cocktail (CJC)/ polyphenols Orange juice (OJ)/Vitamin C Red grape/ polyphenols Intervention/dosage 1.Intervention: Assorted berry products (837 mg) 2. Control: sweet beverage 1. Intervention: 26g cocoa powder + 12g sugar/day (176.2mg) 2. Control: 12g sugar/day Intervention: 1.75g of white chocolate (WC, <1mg /d) 2.75g of dark chocolate (DC, 274mg /d) 3.Dark chocolate enriched with cocoa polyphenols (HPC, 418 mg /d) Intervention: 1. 56mg aglycone isoflavone 2. 90mg aglycone isoflavone 3. 0mg aglycone isoflavone Intervention: ml CJC /(100mg) ml CJC/(200mg) ml CJC/(400mg) Control : placebo juice Invervention: ml OJ/ (128mg) ml OJ/(191mg) ml OJ/(260mg) Intervention: 1.Red wine (M:840 mg, F:560 mg) 2.Red grape extract (M:765mg, F:510mg) 3.Red grape extract-half dose (M:383mg, F:255mg) 4.Water and placebo tablets Results HDL-C in the berry group by 5.2% compared to control HDL-C by 0.31 mmol/l in cocoa group compared to control HDL-C concentration 11.4%, 13.7% in DC and HPC group HDL-C by 2.9% in control group HDL-C by 5.2% in low and 3.6% in high isoflavone group, respectively HDL-C by 4.1% in control group HDL-C by 8.6% (250ml CJC) and 8.1% (500 CJC) compared with placebo juice Consumption of 750ml increased HDL-C by 21% compared with base line HDL-C concentration by 6% in red wine group 55

69 4.4 Effect of Dietary Bioactive Compounds Cholesterol Efflux Excessive cholesterol is removed from lipid loaded macrophages in the sub-intima of the vessel wall and collected by HDL and apoa-1, and thus may potentially suppress the progression of atherosclerosis (107). As more free cholesterol is transferred out of the macrophage, there is less chance for free cholesterol to cause: 1) cell apoptosis and 2) inflammation (106). It is important to conceptualize that to increase the net cholesterol efflux, both the macrophage cholesterol efflux and the capacity of HDL to mediate the efflux should be considered. How bioactive compounds affect both the macrophage cholesterol efflux and capacity of HDL to mediate the efflux has been investigated. Growing evidence has shown that bioactive compounds have positive effects on cholesterol efflux from the macrophage. Data from in vitro studies have shown that resveratrol increased LXR-α mrna expression in both THP-1 and human monocyte-derived macrophages (HMDM). LXR-α regulates the expression of ABCA1, ABCG1 and apoe. Resveratrol caused ABCA1 mrna expression to increase by twofold in THP-1 macrophages (119). Resveratrol also increases ABCA1 and ABCG1 mrna expression in human macrophages which indicates a potential increase in macrophage cholesterol efflux (119). Moreover, the same study reported that resveratrol repressed the expression of the lipid uptake genes, LPL and SR-AII, which may decrease macrophage lipid uptake and prevent foam cell formation (119). Similarly, Voloshyna et al. also demonstrated that resveratrol increased expression of membrane transport protein (ABCAA/G1) in the THP-1 macrophage, thereby increasing apoa-1-mediated cholesterol efflux from macrophages (120). 56

70 Mechanistically, the resveratrol effects on increased transporter protein expression is mediated through the up-regulation of nuclear receptors (LXR-α, PPAR-γ) (120). Voloshyna et al. also reported that resveratrol attenuated lipid accumulation in cultured human macrophages via down regulation of oxldl uptake (120). Taken together, the effects of resveratrol during macrophage cholesterol efflux include: 1) up-regulating nuclear receptor expression and increasing expression of transport protein and 2) attenuating oxldl uptake by cells. Lycopene is a carotenoid found in tomatoes. Palozza et al. investigated the effects of lycopene on the regulation of cholesterol synthesis and efflux in human macrophages (121). Data from the study has shown that lycopene increased ABCA1 expression up-regulated PPAR-γ and LXR-α by 2.2-fold (122). In addition, cav-1 (which is associated with enhanced cholesterol efflux) was up-regulated by 2-fold by lycopene. Lycopene also decreased HMG- CoA reductase expression. These results imply a potential role of lycopene in increasing cholesterol efflux through an increase in membrane transporter protein expression and attenuating cellular cholesterol synthesis. Helal et al. (102) investigated the effect of extra-virgin olive oil (EVOO) on cholesterol efflux. Twelve participants consumed 25ml/d of EVOO for 12 weeks. EVOO consumption increased ABCA1 expression and increased the capacity of human monocytederived macrophages (HMDM) to transfer excess cholesterol to human apoa-1 by over 44% (p<0.001). Helal et al. also evaluated the effect of EVOO on capacity of HDL to accept cholesterol in an ex vivo model. EVOO consumption enhanced the HDL-mediated cholesterol efflux capacity by 11.93% (p<0.05). The improved capacity of HDL to mediate cholesterol efflux is thought to reflect an improvement in HDL function (102). These results indicate that 57

71 consumption of EVOO increases macrophage cholesterol efflux as well as increases HDLmediated cholesterol efflux. Research conducted by Zhang et al. (123) on walnuts and walnut oil demonstrated that ALA or other bioactive compounds reduced SCD1 expression in vitro. These in vitro results reported that walnut oil (0.5 mg/ml) increased cholesterol efflux by 35% in THP-1 macrophage-derived foam cell (MDFC). Walnut oil treated cells did not increase membrane transporter-related gene expression, but walnut oil significantly reduced SCD1 mrna expression (123). The authors further evaluated the postprandial effects of serum collected from subjects fed walnut oil on cholesterol efflux. Fifteen subjects consumed 51g of walnut oil; serum samples were analyzed at baseline and 4h postprandially. An ex vivo model was used to compare baseline and 4 h postprandial serum samples. There was no significant difference in cholesterol efflux between baseline and after four hours. However, SCD1 mrna was greatly reduced after walnut oil consumption. Sub-group results demonstrated an increase in cholesterol efflux by 17% in participants in the low CRP sub-group. Because of a 3-fold increase in ALA in serum after 4 hours of walnut oil consumption, the authors hypothesized that ALA was the primary contributor to SCD1 suppression (123). Later, Zhang et al. (124) investigated the role of ALA on RCT as a potential mechanism to explain the RCT effect. The results revealed that ALA did not affect the cell membrane transporters. Thus, the increased cholesterol efflux with walnut oil was suggested to primarily reflect a decrease in SCD1 expression and the regulation of its target gene (124). Another study conducted by Uto-Kondo et al. (103) examined the effect of coffee consumption on HDL-mediated cholesterol efflux in macrophages. The authors evaluated the effects of phenolic acids from coffee, especially, caffeic acid and ferulic acids on cholesterol 58

72 efflux in vitro, in mice in vivo, and in humans ex vivo. In this study, caffeic and ferulic acids enhanced HDL-mediated cholesterol efflux as well as increased ABCG1 and SR-BI expression in THP-1 macrophages. Subsequently, a crossover study with eight healthy volunteers was conducted. Subjects consumed 350 ml of freshly prepared with 10 g of instant coffee. Blood samples were taken at 0 and 30 mins after coffee consumption. The in vitro and ex vivo data have shown that coffee increased phenolic acids and their metabolites in plasma. Compared to baseline, HDL-mediated cholesterol efflux was increased by 1.4-fold in HMDM after coffee consumption. This was mainly due to the 1.7 fold and 2.5 fold increase in ABCG1 and SR-BI mrna expression, respectively (103). Current evidence suggests that dietary bioactive compounds can improve HDL mediated cholesterol efflux. More clinical investigation is required to evaluate changes in HDL function in response to bioactive compounds consumption. 59

73 Table 4-3. Effects of bioactive compounds on macrophage and HDL-mediated cholesterol efflux Reference Bioactive compounds Macrophage CEx HDLmediating CEx Proposed mechanism(s) Results Sevov et al. (119) Resveratrol Positive Up-regulated LXR-α ABCA1 mrna ABCG1 mrna LPL SR-AII suppresses foam cell formation NA Voloshyna et al. (120) Resveratrol Positive ABCA1 ABCG1 SR-BI + PPARγ Net cholesterol efflux Palozza et al. (121) Lycopene Positive Up regulation of PPARγ, LXR-α thus, ABCA1 Caveolin-1 HMG-CoA reductase Cholesterol efflux Cholesterol synthesis Helal et al. (102) EVOO/polyp henols Positive Positive ABCA1 ABCG1 Macrophage cholesterol efflux Zhang et al. (123) Walnut/ALA Positive Positive SCD1 + FXR, FXR-mediated SCD1 Cholesterol efflux in low CRP sub-group subjects Zhang et al. (124) ALA Positive SCD1 + FXR Cholesterol accumulation in foam cell Uto-Kondo et al. (103) Coffee/ Caffeic acid and ferulic acid + activate, up regulation, down regulation positive HDL-mediated cholesterol efflux via upregulating ABCG1 and SR-BI mrna expression HDL-mediated cholesterol efflux 60

74 4.5 Summary Atherosclerosis is an underlying condition for cardiovascular diseases, some of the most prevalent being myocardial infarction, ischemic stroke and sudden cardiac death. The development of a suitable therapeutic intervention target for atherosclerosis is of great interest. Results from recent pharmaceutical trials have shown that drug therapy that increases HDL-C levels does not reduce CVD morbidity and mortality. Consequently, the current view is that more attention should be paid to improving HDL function. Evidence from preliminary studies suggests that dietary bioactive compounds affect both HDL-C levels and HDL function. The beneficial affect provided by dietary bioactive compounds on cholesterol efflux appears to involve multiple mechanisms, including: 1) up-regulating membrane transporter (i.e., ABCA1, ABCG1, SR-BI) and related gene expression; 2) increasing membrane transporter gene expression (PPARγ, LXR-α, Caveolin-1); and 3) suppressing the key enzyme leading to less cholesterol accumulation in foam cells. With respect to atherosclerosis prevention, both macrophage cholesterol efflux and the capacity of HDL to accept surplus cholesterol should be taken into consideration. More research is required to better understand the effects of bioactive compounds on the capacity of HDL to promote cellular cholesterol efflux. Both dietary intervention and observational studies are needed to further investigate the effect of dietary compounds on HDL functionality, as well as whether improved HDL functionality reduces cardiac events. 61

75 4.6 Objectives and Hypothesis To examine the effects of dietary oils low in SFA and high in MUFA and PUFA on HDL functionality via measuring serum mediated cholesterol efflux in individuals with or at risk for MetS To explore the association between central obesity and HDL functionality by measuring serum mediated cholesterol efflux in individuals with or at risk for MetS Diets low in SFA and with varying unsaturated fatty acid profiles will beneficially affect HDL function by improving serum mediated cholesterol efflux capacity 62

76 Chapter 5 VEGETABLE OILS WITH DIFFERENT UNSATURATED FATTY ACID PROFILES INCREASE SERUM MEDIATED CHOLESTEROL EFFLUX FROM THP-1 MACROPHAGES: THE CANOLA OIL MULTICENTRE INTERVENTION TRIAL 5.1 Abstract Background: Cholesterol efflux (CEx) during reverse cholesterol transport is an important step in preventing the progression of atherosclerosis. Vegetable oils low in saturated fatty acids (SFA) that differ in unsaturated fatty acid profiles favorably affect multiple cardiovascular disease (CVD) risk factors, however, their effects on CEx remain unclear. Objective: The objective of this study was to examine the effects of isocaloric diets containing vegetable oils low in SFA and with varying n-9, n-6 and short and long-chain n-3 fatty acid profiles on serum mediated CEx in individuals with or at risk for MetS. Design: A multi-center, double blind, randomized, 5-period crossover controlled feeding study was conducted. Each diet period lasted 4 wk and was followed by a 4 wk washout. Participants (n=101) with central obesity and at least one other MetS criterion consumed five isocaloric heart healthy diets (50% CHO, 15% PRO, 35% FAT, <7% SFA) containing different treatment oils incorporated in two smoothies/day. The treatment oils 63

77 evaluated were: Canola (62.8% MUFA), CanolaOleic (72% MUFA), CanolaDHA (63.8% MUFA with 5.8% DHA), Corn/Saff (69.3% LA), or Flax/Saff (37.5% LA, 32% ALA). Serum mediated CEx was measured at baseline and at the end of each diet period. Body composition was measured by dual-energy X-ray absorptiometry (DXA) at baseline and the end of each diet period. Results: The Canola, CanolaOleic, CanolaDHA, Corn/Saff and Flax/Saff oil diets increased serum mediated CEx capacity from THP-1 macrophages by 39.1%, 33.6%, 55.3%, 49.2% and 50.7% respectively, compared to baseline (p<0.05 for all). Weight status was found to be an independent predictor of the serum mediated CEx capacity. Participants with a normal BMI had a greater increase in CEx capacity compared with overweight and obese participants. Waist circumference and abdominal adiposity were negatively correlated with serum mediated CEx capacity (r = -0.25, p = 0.012, r = -0.33, p = 0.017, respectively). Conclusion: In individuals with or at risk for MetS risk, consumption of diets low in SFA and enriched with MUFA and/or PUFA improve HDL function by increasing cholesterol efflux capacity. This mechanism may account for additional CVD benefits of diets low in SFA and high in unsaturated fatty acids. 64

78 5.2 Introduction Central obesity is an important risk factor for CVD, in part because of its effects on cardiometabolic risk factors, including high blood pressure, hypertriglyceridemia, hyperglycemia and low HDL-cholesterol (HDL-C). Increasing evidence has shown the atheroprotective effect of HDL depends not only on HDL cholesterol levels, particle size but more importantly on HDL functionality. Recent studies have shown that HDL function (i.e. principally facilitating reverse cholesterol transport) is a better predictor of coronary artery disease (CAD), independent of HDL-C ( ). Previous studies have evaluated the effects of dietary fatty acids on HDL functionality particularly on HDL capacity to mediate CEx (102, 128, 129). Evidence from animal studies has shown that omega-3 fatty acids (EPA and DHA) promote macrophage reverse cholesterol transport (128, 129). Marnillot et al. reported a 79% increase in serum mediated CEx capacity from J744 macrophages in rats after 8 weeks of consuming diets supplemented with omega-3 fatty acids (EPA and DHA, 2.5% of total energy). A similar result was reported by Nishimoto et al. in hamsters fed a diet supplemented with fish oil (8% of total energy) for 4 weeks (128). Evidence from a clinical trial conducted in healthy subjects (n = 26) reported that consumption of extra virgin olive oil (25ml/day) for 12 weeks improved HDL CEx capacity 25% (102). Little is known about the effectiveness of dietary fatty acids on HDL CEx capacity in individuals with MetS. In addition, central obesity, the key characteristic of MetS, is associated with inflammation and oxidative stress which have been shown to modify the 65

79 protective functions of HDL (130). However, the effects of central obesity on HDL function have not been studied extensively. This is particularly relevant to MetS in that a moderate fat that emphasizes unsaturated fatty acids is recommended. The purpose of the present study is to evaluate the effects of diets low in SFA with varying unsaturated fatty acid profiles (i.e. MUFA and PUFA) on serum mediated CEx capacity, and to investigate the association between HDL function and central obesity in a population at risk or with MetS. 5.3 Methods Study Design The Canola Oil Multicentre Intervention Trial (COMIT) was a double blind, randomized, cross-over controlled feeding trial. Detailed methods are published elsewhere (53). In brief, the study included 5 treatment periods. Participants were fed an isocaloric diet and requested to follow their routine physical activity practices to maintain body weight. Weight was measured daily (Monday through Friday) when meals were distributed at each diet center. Each of 5 treatment periods lasted 4 wk and was separated by a 4-wk washout period. One hundred and thirty participants completed the study. A subgroup of participants (n=28) did not receive a metabolically controlled feeding regimen; they prepared their own meals in a communal kitchen under supervision and consumed the smoothies with the treatment oils that were pre-made and delivered. These participants were excluded from the analysis due to the lack of metabolic diet control. One participant whose baseline sample was not available was not included in the analysis. 66

80 5.3.2 Sample Collection Fasting blood samples were collected on days 1 and 2 (baseline) as well as 29 and 30 (endpoint) for all diet periods. Samples from all clinical sites were frozen and shipped to the central lab at the University of Toronto for analysis. Sample collection on day 1 of the study was used as the baseline to calculate the changes in response to study diets Dual-Energy X-Ray Absorptiometry (DXA) Measurements Body composition was assessed at baseline and at the end of each diet period by DXA according to the manufacturer s recommendations (Lunar Prodigy Advance, GE healthcare, Madison, WI, USA; QDR-4500W; Hologic Corp,Waltham, MA ). Total and regional body composition were determined with Prodigy Encore 2005 software (version ) and APEX System software (version 4.0) using their default configurations. Criteria used to identify the anatomical region of interest were consistent across all sites. Baseline body composition was available for a subset of the study population (n=54). All sites performed DXA scans at the end of each diet period (n=101) High Throughput Screen Cholesterol Efflux Assay HumanTHP-1 monocytes were plated in 96-well plates at a density of cells/well and differentiated into macrophages by adding 100 nm phorbol 12-myristate 13-acetate (PMA) for 48 h. After differentiation, cells were washed twice with Phosphate buffered saline (PBS) and cultured in the growth medium overnight. Cells were loaded with 50 μg/ml oxldl and 1μg/ml of 3-NBD cholesterol for 24 h to induce foam cell 67

81 formation and label the intracellular cholesterol pool. After 24 h, cells were washed twice with PBS and incubated with human sera (10%, v:v) for 1 h. After incubation, 100 μl of the medium from each well was collected and transferred to a new 96-well plate. Cells were treated with 100 μl lysis buffer and homogenized on an orbital shaker for 10 min. Fluorescence from the PBS fraction and the cell lysate fraction was measured at excitation and emission wavelengths of 485 and 535 nm, respectively. 5.4 Statistical Analysis Variables were tested for normality and reported as the mean ± standard error. The normality of data was assessed before analysis by using the skewness and the Shapiro- Wilk test and graphically by evaluating histograms. Fit statistics were assessed to identify any outliers (±3 SD). Data not normally distributed were log transformed for analysis. Log data were back transformed for reporting. Statistical analyses were performed using the mixed model procedure in the Statistical Analysis Systems statistical software package version 9.2 (SAS Institute, Cary, NC, US). Mixed-effects repeated-measures analysis of variance was stylized for data analysis. Models included the following factors as fixed effects: diet, period, age, gender, obesity status and baseline values used as fixed effects, participant was used as a repeated factor, with center as random effect. Significant diet effects were determined using Tukey post-hoc tests. Changes from baseline were calculated by subtracting the pre-intervention measurements from the post-intervention measurements. Correlations were performed by Minitab (version 16.2; Minitab). 68

82 5.5 Results Baseline Characteristics Baseline anthropometric and metabolic characteristics of participants are summarized in Table 1. One hundred and one participants (50 males and 51 females) were included in the analyses. Males had a greater body mass (p<0.0001) and waist circumference (p<0.0001) compared with females at baseline. Baseline body composition assessment was conducted in a subgroup of participants (n=54) Table 2. In this subgroup of participants, baseline characteristics were compared with the total study population (n=101); and there were no significant differences in body weight, BMI and other cardiometabolic risk factors, namely, TC, HDL-C, TG, SBP, DBP between these two groups at baseline Diet-Specific Effects on the Lipid/Lipoprotein Profile The lipid/lipoprotein results of participants in COMIT (n=130) have been reported previously (131). Data presented herein are for the individuals (n=101) who only consumed the tightly controlled experimental diets that were prepared in the diet study centers (this excludes the subgroup of participants who prepared their meals in a communal kitchen). As presented in Table 3, all diets decreased TC, LDL-C and TG from baseline (p<0.05 for all) after four weeks of consumption. A significantly greater reduction in TC was observed after consumption of the Flax/Saff oil diet (-0.68±0.043) compared with the CanolaDHA oil diet (-0.54±0.058, p=0.0022). All experimental diets similarly decreased LDL-C from baseline. The CanolaDHA oil diet elicited the greatest 69

83 reduction in TG (-0.5 mmol/l) versus the other test diets (p<0.001 for all comparisons), followed by the Corn/Saff (-0.19±0.047), Flax/Saff (-0.17±0.044), Canola (-0.14±0.05) and CanolaOleic (-0.07±0.044) oil diets. After 4 weeks, HDL-C was higher in response to the CanolaDHA oil diet (0.068±0.016) compared with the Canola (-0.043±0.015, p<0.0001), CanolaOleic (-0.020±0.015, p<0.0001), Corn/Saff ( ±0.015, p<0.0001) and Flax/Saff oil diets (-0.036±0.015, p<0.0001). There were no differences in changes in HDL-C from baseline between five oil diets. The Flax/Saff oil diet led to the greatest reduction in ApoAI compared with the other test diets (p<0.05 for all). There were no differences in changes of ApoAI from baseline in response to Canola, CanolaOleic, CanolaDHA and Corn/Saff oil diets. There was a significantly greater reduction from baseline in ApoB after the Corn/Saff and Flax/Saff oil diets compared with the CanolaDHA oil diet Diet-Specific Effects on Serum Mediated Cholesterol Efflux from THP-1 Macrophages The effect of the different test diets on serum mediated CEx from THP-1 macrophages is presented in Figure 5-1. After 4 weeks, the Canola, CanolaOleic, Canola DHA, Corn/Saff and Flax/Saff oil diets, serum mediated CEx from THP-1 macrophages was increased by 39.1%,33.6%, 55.3%, 49.2% and 50.7%, respectively, from baseline (p<0.05 for all). All diets increased serum mediated CEx capacity similarly. Because there were no treatment differences, participants were pooled on the basis of BMI status. There was an effect of BMI status on serum mediated CEx across the three BMI categories compared to baseline (normal BMI, n=13, p<0.0003; overweight, n=33, p<0.0338; obese, n=53; p<0.0446) Figure 5-2. Participants with a normal BMI had the 70

84 greatest increase (93%) in serum mediated CEx capacity compared with overweight (67%, p=0.036) and obese participants (25%, p=0.031) after diet intervention. There was no significant difference in CEx capacity between overweight and obese participants Correlation Between Waist Circumference, Abdominal Fat Mass and Serum Mediated Cholesterol Efflux at Baseline Baseline correlations between waist circumference (n=101), abdominal fat mass (n=54) and CEx capacity are presented in Figures 5-3 and 5-4. Serum mediated CEx capacity was negatively correlated with waist circumference (r = -0.25, p = 0.012). In a subset of participants who had a body composition assessment conducted at baseline, the capacity of serum to mediate CEx was negatively correlated with abdominal fat mass (r = -0.33, p = 0.017) 5.6 Discussion Our findings demonstrate that consumption of vegetable oils with varying fatty acid profiles (low in SFA and high in unsaturated fatty acids) significantly increases serum mediated CEx capacity in participants either with or at risk for MetS. Weight status assessed by BMI was an independent predictor of serum mediated CEx capacity. Participants with a normal BMI had the greatest increase in CEx capacity compared to overweight and obese participants. Central obesity as assessed by waist circumference and abdominal fat mass was negatively correlated with CEx capacity at baseline. Our findings add to the evidence base that diets high in unsaturated fatty acids beneficially affect CEx capacity. We have shown that diets high in both monounsaturated 71

85 and polyunsaturated fatty acids (both n-6 and n-3 PUFA) increase CEx. Previous studies have reported similar results for omega-3 fatty acids. Marmillot et al. reported a 79% of increase in serum mediated CEx capacity in J744 macrophages in rats after supplementation with omega-3 fatty acids (2.8% of energy) for 8 weeks compared with the control diet (129). Chadli et al. demonstrated that hamsters fed an omega-3 fatty acid (EPA)-enriched high fat diet (21% fat w/w, 4.5% of energy as EPA) for 20 weeks had a higher CEx (31.9%) from Fu5AH cells compared to a high fat (21% fat w/w) and control diet (5% fat w/w) (132). In addition, Pal and Davis have shown that EPA (60 μg/ml) incubated with human fibroblasts enhanced CEx (133). The main function of HDL is to maintain cholesterol homeostasis by removing excess cholesterol from peripheral cells and transferring it to the liver to be metabolized and excreted. This process involves the interaction of lipid-poor apo-ai and mature HDL with ABCA1/G1 and SR-BI receptors, respectively. The increase in serum mediated CEx reported herein suggests an improved HDL functionality. Serum mediated CEx is dependent on the chemical properties of HDL. With respect to this, HDL phospholipid (PL) composition (not measured in the present study) is affected by long chain PUFA (n3 and n6) (134, 135). The fact that both linoleic acid and alpha-linolenic acid cannot be synthesized in humans indicates that the lipid membrane FA composition reflects the abundance of these PUFA in the diet (135, 136). This has been confirmed by Davidson et al. using reconstituted HDL that varied in PL fatty acids; HDL particles with a fluid surface rich in unsaturated fatty acids were capable of accepting free cholesterol at a faster rate and greater amount than HDL enriched in long chain SFA (137). 72

86 In the present study, the increase in CEx capacity was observed after consumption of two MUFA enriched diets (Canola, 39.1%; CanolaOleic 33.6%). Sakr et al. reported that consumption of high-oleic sunflower oil (73% MUFA) for four consecutive days increased CEx from Fu5AH cells as a result of an enrichment in small dense HDL 3 PL (138). Sola et al. reported that consumption of a diet enriched with olive oil (15.6% of total energy) for 7-week increased the capacity of HDL 3 to promote CEx as a result of physiochemical changes in HDL 3 (greater fluidity, higher cholesteryl ester content, and smaller size) (139). Helel et al. also found that consumption of extra virgin olive oil (71% MUFA) for 12-weeks increased the capacity of serum to mediate CEx from THP-1 macrophages by 9.8% (p<0.01) as the result of a 13% increase in the fluidity of the HDL PL layer (102). In the present study, in which all diets were low in SFA (<7%), it is likely that there was an increase in the surface fluidity of HDL particles which would be expected to enhance CEx capacity. In addition to the beneficial effect of accepting free cholesterol, previous studies have demonstrated that both PUFAs and MUFAs up-regulate transport protein (ABCA1/G1) gene expression (102, 123, 124). Further research is needed to better understand the underlying mechanisms that account for the effects of dietary FAs on CEx from peripheral tissues. Obesity typically decreases plasma HDL-C levels and also affects HDL structure which likely impairs HDL function (140). Similar to previous findings (141, 142), we observed a greater increase in serum mediated CEx in participants with a normal BMI compared with overweight and obese participants. In addition, we reported a negative correlation between waist circumference, abdominal fat mass and serum mediated CEx capacity at baseline in participants with or at risk for MetS. Sasahara et al. have reported 73

87 that overweight/obese subjects had impaired serum CEx capacity from fibroblasts (142). Autran et al. similarly reported a reduced serum CEx capacity in obese women (143). Obesity is associated with increased inflammation and oxidative stress that could contribute to an impaired HDL functionality and consequent diminution of CEx (144, 145). A high BMI is negatively associated with the anti-oxidant capacity of HDL (146). The greater serum mediated CEx capacity in normal BMI participants is suggestive of an impairment in HDL functionality in overweight and obese individuals. The correlation between central obesity and CEx demonstrates a metabolic link between adipose tissue and HDL function. There is a shift in focus from quantifying HDL-C to assessing changes in HDL function in the field. Our study demonstrates the importance of assessing HDL function rather than just HDL-C levels. A strength of the present study is the controlled study design, including the diet design which was tightly controlled. The treatment oils were delivered via smoothies for easy and accurate administration. In addition, the cross-over study design minimized the influence of any genetic polymorphisms that contribute to variations in diet responsiveness and consequent inter-individual differences in results among study participants (77). Potential limitations of the study include lack of baseline body composition data for all participants resulting in less statistical power to further explore the relationship between body weight and/or abdominal fat mass changes with CEx. 74

88 5.7 Conclusion In summary, short-term consumption of diets low in SFA and with different unsaturated fatty acid profiles improved serum mediated CEx in participants with or at risk for MetS. The present study underscores the adverse effects of overweight/obesity on CEx. Importantly, our findings add to the evidence in support of the beneficial effect of diets low in SFA and high in unsaturated fatty acids for reducing cardiometabolic risk that we suggest are mediated, in part, by an increase in HDL functionality as measured by CEx. With weight loss and maintenance of an ideal body weight, overweight and obese individuals can benefit from a heart healthy diet low in SFA and high in unsaturated fatty acids that promotes CEx. 75

89 Table 5-1. Baseline anthropometric and cardiometabolic characteristics Characteristic Anthropometric measurements All participants (n=101) Males (50) Females (51) Age 49.5± ± ±1.5 Body mass (kg) 85.8± ±1.9 * 75.9±2.1 * Height (m) 1.7± ± ±0.01 Body mass index (kg/m2) 29.4± ± ±0.6 Waist circumference 101.9± ±1.2 * 96.8±1.4 * Metabolic syndrome risk factors Glucose (mmol/l) 5.24± ± ±0.20 HDL-cholesterol (mmol/l) 1.25± ± ±0.05 Triglycerides (mmol/l) 1.84± ± ±0.12 Blood pressure SBP/DPB (mmhg) 122/78 123/78 118/78 Values are expressed as means ± SEM * Values were significantly different between gender p<

90 Table 5-2. Body composition of participants at baseline All participants (n=54) Males (n=20) Females (n=34) Body fat mass (kg) 32.9± ± ±1.5 Body lean mass (kg) 51.3± ±2.0 * 43.3±0.9 Trunk fat mass (kg) 17.2± ±1.1 * 16.4±0.8 Trunk lean mass (kg) 24.9± ±1.1 * 21.1±0.5 Android fat mass (kg) 3.1± ±0.2 * 2.9±0.2 Android lean mass (kg) 3.7± ±0.1 * 3.1±0.08 Gynoid fat mass (kg) 5.6± ± ±0.3 Gynoid lean mass (kg) 7.9± ±0.3 * 6.7±0.2 Values are expressed as means ± SEM * Values were significantly different between gender p<

91 Table 5-3. Changes of serum lipid profiles from baseline in response to experimental diets Baseline Canola Oil CanolaOleic CanolaDHA Corn/ Saff Flax/ Saff TC (mmol/l) 5.48± ±0.051* a -0.61±0.056* a -0.54±0.058* ab -0.62±0.057* a -0.68±0.043* ac HDL-C (mmol/l) 1.22± ±0.015 a ±0.015 ad 0.068±0.016 b ±0.015 a ±0.015 cd LDL-C (mmol/l) 4.26± ±0.048* -0.55±0.05* -0.58±0.054* -0.59±0.053* -0.60±0.048* TG (mmol/l) 1.75± ±0.05* a -0.07±0.044* a -0.5±0.044* b -0.19±0.047* a -0.17±0.044* a Apo-AI (mmol/l) 1.47± ±0.013 a ±0.012 a ±0.012 a ±0.013 a ±0.012* b Apo-B (mmol/l) 1.06± ±0.011 a -0.11±0.012 a -0.10±0.012 ac -0.13±0.012 ad -0.12±0.011 ad Values are expressed as means ± SEM Values in a row with different superscript letters indicate significant differences between diets p<0.05 (Mixed model post hoc analysis) * Values were significantly different from baseline p<

92 Cholesterol efflux capacity (% change from baseline) Figure 5-1. Changes in the CEx capacity in response to the test diets * * * 40 * * 20 0 Canola CanolaOleic CanolaDHA Corn/Saff Flax/Saff 79

93 Cholesterol efflux capacity (% change from baseline) Figure 5-2. Changes in the CEx capacity in response to BMI status 120 * * 60 * Normal Overweight Obese 80

94 Baseline Cholesterol Efflux (%) Figure 5-3. Correlation between waist circumference and CEx capacity at baseline for all participants (n=101) Baseline Waist Circumference (cm) 81

95 Baseline Cholesterol Efflux (%) Figure 5-4. Correlation between abdominal fat mass and CEx capacity at baseline in a subgroup of participants (n=54) Baseline Android Fat Mass (g) 82

96 Chapter 6 MEASUREMENT OF ABDOMINAL ADIPOSE TISSUE IN A SUBSET OF COMIT PARTICIPANTS: A COMPARISON of MRI, DXA and ANTHROPOMETRIC MEASUREMENT 6.1 Abstract Introduction Subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) differ metabolically. In many studies, excessive VAT is linked to inflammation and obesity-related diseases. Objective We evaluated the cross-sectional associations between baseline abdominal SAT mass, VAT mass, CRP level and anthropometric measurements in a subgroup of subjects from the Canola Oil Multicentre Intervention Trial (Females = 7; Males = 7) with increased waist circumference (Female: >80cm; Male: >94cm) and at least one other metabolic syndrome risk factor. MRI images were obtained from the lower abdomen region: one slice of the image below and nine slices of the image above the center of lumbar vertebrae L4-5 were obtained. 83

97 Results Regression analysis demonstrated that at baseline, in both males and females, CRP was positively correlated with VAT mass (r = 0.92, p < ). Waist circumference was positively correlated with SAT mass (r = 0.59, p = 0.034). In addition, BMI was positively correlated with SAT mass (r = 0.83, p = ). There was no correlation between: CRP level and SAT mass; waist circumference and VAT mass; and BMI and VAT mass. Conclusion In summary, in males and females with abdominal obesity, CRP was positively correlated with VAT mass, but not SAT mass. Waist circumference and BMI were positively correlated with SAT mass. CRP levels can be used as an indicator of visceral fat mass in subjects with at least two metabolic syndrome criteria, one of which is increased waist circumference. Waist circumference was a better predictor of subcutaneous fat mass. Clinical measurements of CRP, BMI and waist circumference provide insight on types of fat deposition in the abdominal area in patients at risk for metabolic syndrome with increased waist circumference. 6.2 Introduction Metabolic syndrome (MetS) is characterized by increased WC and at least two other risk factors that include insulin resistance, dislipidemia and hypertension. Various anthropometric indices have been used to assess abdominal adiposity to further predict cardiometabolic risk. In clinical practice, WC is a surrogate marker which is used to assess 84

98 abdominal obesity. Emerging evidence has suggested that in addition to the mass of abdominal adipose tissue, the types of abdominal adipose tissue is also important for cardiometabolic risk (147). Abdominal fat is composed of abdominal subcutaneous fat and visceral fat. Previous studies have shown that excess visceral adipose tissue (VAT) is associated with inflammation, insulin resistance and the development of diabetes ( ). In the Dallas Heart Study, a higher VAT mass at baseline was independently associated with diabetes; specifically, an increase of 1.4kg VAT mass was associated with a 2.4-fold increase in developing type 2 diabetes (151). Sironi et al. reported male participants with hypertension and insulin resistance had more VAT (152). In contrast, the subcutaneous adipose tissue depot (SAT) serves as a storage reservoir that is less metabolic activity (153, 154). Crude anthropometric measures such as WC offer an overall assessment of central obesity, but are inadequate to quantify adipose tissue mass in specific depots, and how these are related to cariometabolic risk. Accurate measurement of abdominal adipose tissue is fundamental to detect changes in adipose tissue distribution. Recent body-imaging technology has been applied in clinical practice to examine abdominal adiposity. MRI offers the most accurate assessment of VAT and SAT. Due to its limited availability and cost, MRI is not commonly used clinically. DXA is becoming increasingly popular for the measurement of body composition as well as the quantification of VAT and SAT in the abdominal region. There is limited information that has validated SAT and VAT measurements obtained using DXA versus MRI. 85

99 The purpose of the present study was 1) to evaluate the association between VAT, SAT mass and anthropometric measurements in a subgroup of participants from COMIT, and 2) to compare two noninvasive methods (MRI, DXA) for estimating abdominal adipose tissue distribution. 6.3 Materials and Methods The study was a multi-center, double blind, randomized, cross-over controlled feeding trial. Detailed methods are published elsewhere (53) Anthropometric Measurements Waist circumference was measured according to the NHLBI guidelines. Participants were in a standing position. To measure WC, an inelastic measuring tape was placed in a horizontal plane around the abdomen at the hip bones that are located on top of the right iliac crest. Two measurements were obtained. Measurements were made by two certified nurse practitioners DXA Measurements Body composition was assessed pre-intervention (baseline) and the end of each diet period (day 29) by DXA according to the manufacturer s recommendations (Lunar Prodigy 86

100 Advance, GE healthcare, Madison, WI, USA; QDR-4500W; Hologic Corp,Waltham, MA ). Total and regional body composition including abdominal VAT and SAT fat mass were determined with Prodigy APEX System software (version 4.0) using their default configurations MRI A subset of participants (n=14) from The Pennsylvania State University study site was offered an MRI scan. Participants received one MRI scan at the beginning of the study and one scan after each diet period. Screening forms were filled out before entering the scanning room at each visit. Participants were required to remove any metal objects and wear scrubs. All MRI data were acquired with a Siemens Magnetom Trio 3.0 scanner equipped with a body coil. Participants were positioned in the magnet in a supine position, arms placed laterally. Each scan routine includes 24 single-slice axial abdominal fat spin echo images acquired (TR/TE=100/2.46ms; flip angle=70 o, number of excitation = 1; bandwidth=280 khz, matrix=456x192; slice thickness = 8mm; echo train length = 4). Acquisition was centered on vertebral disc L3 (12 slices above, 12 slices below) Adipose Tissue Quantification A program we developed (Matlab; MathWorks, Natick, MA) was used to quantify VAT and SAT mass. We analyzed the sum of a total of 10 axial image slices starting from one image slice below the center of L4-5 (Figure 6-1). The 10 image slices cover the entire 87

101 lower abdominal region (from L4-5 to L3). Once the imaging slices were selected, the outer boundary around SAT was automatically displayed (green line) Figure 6-2. Two trained technicians manually traced around the inside boundary of SAT (yellow line) and the outside edge of VAT (red line); for scans with >5% variation between technicians the traces were redone. Voxel counts of the white areas were presented from SAT and VAT segments, separately. An average of the voxel count from both technicians was calculated. Adipose tissue mass was calculated as: voxel counts*0.028(voxel size)*0.916g/cm 3 (density of adipose tissue). Figure 6-1. Axial slices selection L3 88

102 Figure 6-2. Segmentation method 6.4 Statistical Methods Statistical analyses were performed by SAS software (version 9.2; SAS institute Inc.) Differences in variables between treatments and genders were analyzed by ANOVA. All significance tests were two tailed and conducted at the 5% significant level. Spearman rank correlations between the MetS risk factors and central obesity assessed by DXA and MRI were examined at baseline. To determine the normality of the data, the skewness of the residuals from the final models were assessed. Differences were considered significant at p 0.05 and as tendencies at p

103 6.5 Results Fourteen of 29 participants from the PSU study site agreed to participate in the optional MRI scans. Baseline characteristics of the participants are presented in Table 6-1. Baseline body composition is presented in Table Participant Characteristics Baseline anthropometric and metabolic characteristics of participants are summarized in Table 6-1. In fourteen subjects, males had a greater body mass (102.1kg vs. 80.2kg, p=0.0038) compared with females at baseline. Participants maintained a consistent level of physical activity throughout the study (data not shown). There were no significant differences in BMI, waist circumference, glucose, TG, DBP and SBP between genders at baseline. As expected, females had a higher HDL-C than males (p=0.0041) at baseline. There were no significant differences in SAT and VAT mass between genders. Interestingly, there was a tendency for females to have a greater ratio of VAT to SAT compared with males (p=0.07). The VAT to SAT ratio is a metric of fat distribution. An increased VAT to SAT ratio suggests a greater VAT mass which indicates increased cardiometabolic risk. Body composition at baseline is presented in Table 6-2. There were no significant differences in body fat mass, trunk fat mass, android fat mass and gynoid fat mass between males and females. Males had a significantly greater body lean mass, percent of body lean mass, trunk lean mass and android lean mass (p<0.05 for all) compared with females. Females had a significantly higher percent body fat mass (43.82% vs %) compared with males (p=0.0014). 90

104 6.5.2 Correlations between VAT, SAT Mass and Anthropometric Measurements Baseline correlations between VAT mass, SAT mass, body composition, and cardiometabolic risk factors are presented in Table 6-3. SAT mass was positively correlated with waist circumference (r=0.59, p=0.034), BMI (r=0.83, p=0.0008), total fat mass (r=0.86, p=0.0002), percent fat mass (r=0.59, p=0.035) and abdominal fat mass (r=0.85, p=0.0002). VAT mass was positively correlated with total fat mass (r=0.64, p=0.018), percent fat mass (r=0.62, p=0.017) and android fat mass (r=0.73, p=0.005). Baseline CRP level was positively correlated with VAT mass (r=0.92, p<0.0001). There was no significant correlation between SAT mass with CRP at baseline Gender Differences in Correlations between VAT, SAT Mass and Anthropometric Measurements Males and females have different adipose tissue depot distributions. A sub-analysis demonstrated that abdominal fat mass was positively correlated with VAT mass in males (r=0.93, p=0.02) but not in females; abdominal fat mass was positively correlated with SAT mass in females (r=0.86, p=0.05) Figure 6-3.A) and B). There was no correlation between abdominal fat mass and SAT in males. Waist circumference was positively correlated with VAT mass (r=0.75, p=0.0051) in males. In contrast, waist circumference was positively correlated with SAT mass in females (r=0.84, p=0.017) Figure 6-4.A) and B) Comparison between MRI and DXA in VAT, SAT Measurements The second aim of this study was to compare different noninvasive methods of estimating abdominal fat distribution in individuals with or at risk for MetS. Results showed 91

105 there was a strong positive correlation for VAT mass (r = 0.94, p<0.0001) Figure 6-5. and SAT mass (r=0.92, p<0.0001) Figure 6-6. obtained using DXA and MRI in this subgroup of participants from COMIT. 6.6 Discussion In this subgroup of COMIT participants, SAT mass was positively correlated with anthropometric measurements including WC, BMI, total fat mass, percent fat mass and abdominal fat mass. VAT mass was positively correlated with CRP but not SAT mass. Increased VAT mass was adversely associated with increased cardiometabolic risk in overweight and obese participants (155). At baseline, although male participants had a greater body weight, there was no significant difference in VAT between male and female participants. Previous studies have shown that there are gender differences in adipose tissue deposition with males having greater VAT deposition, whereas females have more SAT (156, 157). In the present study, the lack of difference between genders in VAT mass could due to the significant age difference for male and female participants. Visceral adipose tissue increases with age (158). In this cohort, female participants were older than male participants which could contribute to the discrepancy. The ratio of VAT to SAT is also strongly related with disorders of glucose and lipid metabolism in obese subjects. It has been shown that individuals with a VAT to SAT ratio greater than 0.4 have significantly more metabolic disorders (147). The tendency of female participants to have a greater VAT to SAT ratio (p=0.07) suggests a disproportional distribution between VAT and SAT, which indicates a potential increased cardiometabolic risk. 92

106 Compared to VAT, SAT was strongly associated with more anthropometric measurements such as WC, BMI and total and regional fat mass. VAT was strongly associated with an inflammation marker. Faber et al. reported that visceral fat mass thickness measured by ultrasonnography was the strongest contributor to CRP concentrations in patients with cardiovascular diseases (159). In Japanese subjects with mild obesity, visceral fat as assessed by computer tomography was a strong and independent predictor of CRP (160). As mentioned, WC is the most commonly used anthropometric measurement to determine central obesity. Data from the present study showed that WC as a surrogate measure provides limited information on adipose tissue distribution. The second aim of the study was to compare two noninvasive body imaging methods for estimating abdominal VAT and SAT distribution in obese subjects. The results showed that VAT and SAT obtained by DXA were strongly correlated with VAT and SAT assessed by MRI. Despite the small sample size of the present study, this significant correlation between these two methods indicates that segmentation of VAT and SAT by DXA can be used as a practical approach in a clinical setting. A limitation of this study is that it was an exploratory subgroup analysis and, therefore, was not powered to detect differences in VAT loss in response to diets. Another possible limitation is that we did not use a commercial program to analyze the MRI data. The approach of two independent, blinded technicians who manually traced image scans was tightly regulated to ensure consistency; however, errors may have occurred. In summary, in males and females with abdominal obesity, CRP was positively correlated with VAT mass, but not SAT mass. Waist circumference and BMI were positively 93

107 correlated with SAT mass. CRP levels can be used as an indicator of visceral fat mass in subjects with or at risk for MetS. Waist circumference was a better predictor of subcutaneous fat mass. Clinical measurements of CRP, BMI and waist circumference provide insight about the types of fat deposition in the abdominal area in individuals at risk for MetS with an increased waist circumference. 94

108 Table 6-1. Baseline anthropometric and cardiometabolic characteristics Males (n=7) Females (n=7) p-value Anthropometric assessment Age (yrs) 37.3 ± 3.9* 55.4 ± 2.2 < Weight (kg) ± 4.3* 80.2 ± BMI (kg/m 2 ) 31.3 ± ± 1.9 NS Waist Circumference (cm) ± ± 1.3 NS Metabolic syndrome risk factors Glucose (mmol/l) 5.2 ± ± 0.2 NS HDL-cholesterol (mmol/l) 0.9 ± 0.1* 1.4 ± Triglycerides (mmol/l) 2.1 ± ± 0.3 NS DBP/SBP (mmhg) 83/125 81/117 NS Adipose tissue mass SAT 2.4 ± ± 0.2 NS VAT 1.0 ± ± 0.09 NS VAT/SAT ratio 0.4± ± Values are expressed as means ± SEM * Values were significantly different between gender p<0.05 NS: non-significant 95

109 Table 6-2. Baseline body composition Males (n=7) Females (n=7) p-value Body fat mass (kg) 31.6± ±1.5 NS Body lean mass (kg) 70.5±1.4* 44.6± Body fat mass (%) 30.9±0.9* 43.8± Body lean mass (%) 69.1±0.9* 56.2±0.9 < Trunk fat mass (kg) 17.3± ±0.8 NS Trunk lean mass (kg) 34.3±0.7* 22.5±0.4 < Abdominal fat mass (kg) 3.2± ±0.2 NS Abdominal lean mass (kg) 4.8±0.1* 3.3±0.1 < Gynoid fat mass (kg) 5.1± ±0.3 NS Gynoid lean mass (kg) 11.0±0.2* 7.0±0.1 < Values are expressed as means ± SEM * Values were significantly different between gender p<0.05 NS: non-significant Table 6-3. Spearman correlations of baseline SAT, VAT and body composition measurements SAT VAT r p-value r p-value Body weight 0.39 NS NS Waist circumference BMI Total fat mass Percent fat mass Abdominal fat mass A/G ratio 0.09 NS 0.31 NS NS: non-significant 96

110 Table 6-4. Spearman correlations of baseline SAT, VAT and cardiometabolic biomarkers SAT VAT r p-value r p-value Glucose 0.41 NS 0.37 NS Triglycerides NS NS HDL-C 0.21 NS 0.51 NS DBP NS 0.18 NS SBP NS 0.34 NS CRP 0.18 NS 0.92 < CHOL NS NS ApoB NS NS ApoAI 0.29 NS 0.38 NS NS: non-significant Table 6-5. VAT and SAT mass in response to five diets Adipose Tissue Mass (kg) Canola CanolaOleic CanolaDHA Corn/Saff Flax/Saff Visceral 1.0± ± ± ± ±0.1 Subcutaneous 2.5± ± ± ± ±0.2 97

111 Abdominal fat mass (kg) Abdominal fat mass (kg) Figure 6-3. A) Correlation between abdominal fat mass and VAT; B) Correlation between abdominal fat mass and SAT A Female Male r=0.62, p= r=0.93, p= VAT mass (kg) B 8.0 Female Male r=0.86, p= r=0.70, p= SAT mass (kg) 98

112 Waist Circumference (cm) Waist Circumference (cm) Figure 6-4. A) Correlation between WC and VAT mass; B) Correlation between WC and SAT mass A 140 Female Male 120 r=0.75, p=0.051 r=0.47, p= B VAT mass (kg) 140 Female Male r=0.46, p=0.29 r=0.84, p= SAT mass (kg) 99

113 Figure 6-5. Correlation between MRI obtained and DXA obtained VAT mass 100

114 Figure 6-6. Correlation between MRI obtained and DXA obtained SAT mass 101