The Pennsylvania State University The Graduate School College of Health and Human Development EFFECTS OF DIETS ENRICHED IN CONVENTIONAL AND HIGH-OLEIC ACID CANOLA OILS COMPARED TO A WESTERN DIET ON LIPIDS AND LIPOPROTEINS, GENE EXPRESSION, AND THE GUT ENVIRONMENT IN ADULTS WITH METABOLIC SYNDROME FACTORS A Dissertation in Nutritional Sciences by Kate Joan Bowen © 2018 Kate Joan Bowen Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2018 The dissertation of Kate Joan Bowen was reviewed and approved* by the following: Penny Kris-Etherton Distinguished Professor of Nutritional Sciences Dissertation Advisor Chair of Committee Gregory Shearer Associate Professor of Nutritional Sciences Sheila West Professor of Biobehavioral Health Peter Jones Distinguished Professor of Human Nutritional Sciences and Food Sciences University of Manitoba Special Member Lavanya Reddivari Assistant Professor of Food Science Purdue University Laura E. Murray-Kolb Associate Professor of Nutritional Sciences Professor-in-Charge of the Graduate Program *Signatures are on file in the Graduate School ii ABSTRACT The premise of this dissertation was to investigate the effects of diets that differed only in fatty acid composition on biomarkers for cardiovascular disease (CVD) in individuals with metabolic syndrome risk factors, and to explore the mechanisms underlying the response. In a multi-site, double blind, randomized, controlled, three period crossover, controlled feeding study design, participants were fed an isocaloric, prepared, weight maintenance diet plus a treatment oil for 6 weeks with washouts of ≥ 4 weeks between diet periods. The treatment oils included conventional canola oil, high-oleic acid canola oil (HOCO), and a control oil (a blend of butter oil/ghee, flaxseed oil, safflower oil, and coconut oil). The oils provided approximately 18% of total energy (60 g per 3000 kcal) and were consumed daily in a strawberry orange smoothie. The macronutrient profiles of the three test diets were as follows: canola oil diet [17.5% monounsaturated fatty acid (MUFA), 9.2% polyunsaturated fatty acid (PUFA), 6.6% saturated fatty acid (SFA)], HOCO diet (19.1% MUFA, 7.0% PUFA, 6.4% SFA), and control diet (10.5% MUFA, 10.0% PUFA, 12.3% SFA). The control diet was formulated to emulate contemporary Western fatty acid intakes. In the first study, we examined the effects the three diets on endpoint lipids, lipoproteins, and apolipoproteins in 119 participants. After 6 weeks, the canola oil and HOCO diets resulted in lower circulating total cholesterol (-4.2% and -3.4%, respectively; P<0.0001), low-density lipoprotein-cholesterol (LDL-C; -6.6% and -5.6%; P<0.0001), apolipoprotein (apo) B (-3.7% and -3.4%; P=0.002), and non-high-density lipoprotein-cholesterol (non-HDL-C; -4.5% and - 4.0%; P=0.001) compared to the control diet. There were no differences between the two canola oil-based diets in these blood parameters. The total cholesterol: HDL-C and apo B: apo A1 ratios iii were lower after the HOCO diet compared to control (-3.7% and -3.4%, respectively). No diet effects on triglyceride, HDL-C, or apo A1 levels were observed. In the second study, we aimed to investigate the biological mechanisms underlying the lipid and lipoprotein response to the treatments, and assessed the expression of 17 target genes that regulate lipid and lipoprotein metabolism. RNA was extracted from peripheral blood mononuclear cells (PBMC) in a subset of the study participants (n=42) and PCR was utilized to determine the relative mRNA transcript at the endpoint of each diet. Relative to the control diet, the canola oil and HOCO diets decreased expression of the ATP-binding cassette (ABC) transporters A1 (canola: -16%, P=0.0006; HOCO: -11%, P=0.005) and G1 (canola: -9%, P=0.003; HOCO: -15%, P=0.003). No significant treatment effects on expression of the remaining genes were observed. The expression of ABCA1 and ABCG1 was not correlated with endpoint circulating lipids, lipoproteins, or apolipoproteins. In the third study, we investigated an additional potential mechanism driving the cholesterol-lowering response to the canola oil-based diets versus the Western diet, and assessed the effects on fecal short-chain fatty acid (SCFA) levels. Fecal samples were collected in a subset of participants (n=20), and SCFA were extracted and quantified using gas chromatography-mass spectrometry. After 6 weeks, a trend toward a treatment effect on endpoint propionic acid was observed (P=0.09). Acetic acid was increased from baseline following the control diet (P=0.04). After the control diet only, fecal levels of propionic acid were positively correlated with blood levels of LDL-C, non-HDL-C, and apo B (rP =0.52 to 0.64, P=0.003 to 0.02), and acetic acid was positively correlated with LDL-C and apo B (rP =0.48 to 0.49, P=0.03 to 0.04). No significant correlations between fecal SCFA and lipids and lipoproteins were observed after the two canola oil-based diets. iv Overall, these data indicate that conventional canola oil and HOCO-based diets elicit comparable, beneficial effects on biomarkers consistent with CVD risk reduction compared to a diet with a Western fatty acid profile. We investigated the underlying cardiovascular health promoting mechanisms in two exploratory analyses, and report evidence of coordinated shifts in genes that regulate cholesterol and phospholipid homeostasis in PBMC, as well as hypothesis- generating data to suggest that the gut environment may mediate the effects of dietary fatty acids on the lipid response. Together, these three studies provide information to add to the existing knowledge of the role of dietary fatty acids in modulating cholesterol homeostasis in humans. v TABLE OF CONTENTS List of Figures .............................................................................................................................. viii List of Tables ................................................................................................................................. ix Abbreviations .................................................................................................................................. x Acknowledgements ....................................................................................................................... xii Chapter 1: Introduction ................................................................................................................... 1 Chapter 2: Literature review ........................................................................................................... 4 2.1. Dietary fatty acids: Structure, sources, and intakes ............................................................. 4 2.2. USFA as a replacement for SFA .......................................................................................... 5 2.2.1. Dietary recommendations for SFA ............................................................................... 5 2.2.2. Macronutrient substitution ............................................................................................ 6 2.2.3. Vegetable oils to increase USFA intake ..................................................................... 10 2.3. Dietary canola oils ............................................................................................................. 11 2.3.1. Conventional canola oil .............................................................................................. 11 2.3.2. HOCO ......................................................................................................................... 13 2.4. Cardiovascular health benefits of canola oils .................................................................... 17 2.4.1. Cardiovascular outcomes ............................................................................................ 17 2.4.2. Atherogenic lipid and lipoprotein biomarkers ............................................................ 21 2.5. Potential mechanisms by which canola oil improves lipids/lipoproteins .......................... 30 2.5.1. Gene expression .......................................................................................................... 30 2.5.2. Phytosterols ................................................................................................................. 34 2.5.3. Gut microbial metabolites ........................................................................................... 35 2.6. Rationale for current research ............................................................................................ 40 2.7. Objectives and hypotheses ................................................................................................. 41 Chapter 3: Diets enriched with conventional or high-oleic acid canola oils decrease atherogenic lipids and lipoproteins compared to a diet with a Western fatty acid profile in individuals at risk for MetS ........................................................................................................................................ 43 Abstract ..................................................................................................................................... 43 Introduction ............................................................................................................................... 45 Methods..................................................................................................................................... 46