Search

Genome-wide association studies have greatly improved our understanding of the contribution of common variants to the genetic architecture of complex traits. However, two major limitations have been highlighted. First, common variant associations typically do not identify the causal variant and/or the gene that it is exerting its effect on to influence a trait. Second, common variant associations usually consist of variants with small effects. As a consequence, it is more challenging to harness their translational impact. Association studies of rare variants and complex traits may be able to help address these limitations. Empirical population genetic data shows that deleterious variants are rare. More specifically, there is a very strong depletion of common protein truncating variants (PTVs, commonly referred to as loss-of-function variants) in the genome, a group of variants that have been shown to have large effect on gene function, are enriched for severe disease-causing mutations, but in other instances may actually be protective against disease. This thesis is divided into three parts dedicated to the study of protein truncating variants, their medical relevance, and their functional consequences. First, I present statistical, bioinformatic, and computational methods developed for the study of protein truncating variants and their association to complex traits, and their functional consequences. Second, I present application of the methods to a number of case-control and quantitative trait studies discovering new variants and genes associated to breast and ovarian cancer, type 1 diabetes, lipids, and metabolic traits measured with NMR spectroscopy. Third, I present work on improving annotation of protein truncating variants by studying their functional consequences. Taken together, these results highlight the utility of interrogating protein truncating variants in medical and functional genomic studies.

Cholesterol equilibrium is meticulously orchestrated, and its proper regulation is pivotal inmitigating the risk of cardiovascular disorders. To preserve this delicate balance,cholesterol must activate sensing mechanisms at organelle membranes within the cell’sinterior. The recently discovered Aster protein family (Aster-A, -B, and -C) partakes innonvesicular cholesterol import to organelles from the plasma membrane (PM). Despitetheir high coding sequence homology, each Aster protein exhibits distinct tissue expressionpatterns, conferring unique functions. This work elucidates their nuanced roles inregulating whole-body cholesterol flux. It scrutinizes how existing therapeutics modulatecholesterol flux, emphasizing their effects on key proteins that are known regulators ofcholesterol homeostasis. The introductory chapter reviews the intended and off-targeteffects of these existing therapeutics. Aster proteins are then introduced as novelmodulatory nodes in cholesterol regulation. Aster-C, prominently expressed in sterol-regulatory tissues like the liver and testis, presents an intriguing target, especially inpotential therapeutic applications for dyslipidemia-associated disease pathology. The bodychapter delves into Aster-C’s role in whole-body homeostasis, unveiling its contribution tocholesterol balance and revealing modest effects on bile acid metabolism under low-cholesterol dietary conditions. Aster-C knockout prompts upregulation in the messengerribonucleic acid (mRNA) levels of paralogs Aster-A and -B in murine liver tissue,suggesting nuanced roles and potential redundancy with other Aster proteins. Comparative nalysis with existing literature indicates that Aster-C’s hepatic transcription is inducibleby FXR stimulation. These findings, intersected with our own research on the Aster-Cknockout-induced disruption of bile acid homeostasis, suggest an integral role for Aster-Cin mediating the rate of reverse cholesterol transport within hepatic tissue. Overall, wedeconstruct cholesterol metabolism, identify existing therapeutic interventions, and thenpropose potential benefits of targeting Aster proteins as novel nodes by which the rate ofreverse cholesterol transport can be stimulated. Ultimately, we aim to explore noveltherapeutic applications in Aster modulation by potentiating clinical relevance in our futuredirections section. Herein, we hope to broaden perspectives on inter-tissue cholesterolhomeostasis dynamics by emphasizing the need for further investigations into the role ofAster proteins in cholesterol trafficking.

Two of the top killers of the human race are cardiovascular disease and type 2 diabetes. These diseases, which have shared etiology, collectively form the domain of cardiometabolic health. Multiple functions of cardiometabolic health are perturbed by impaired sleep, and conversely, restored by increased sleep quantity and/or improved sleep quality. That is, impairments in sleep are closely related to impairments in cardiometabolic health. This leads to the proposition that specific features of sleep act as biomarkers for specific features of cardiometabolic health. This thesis aims to determine the impact of sleep on three major domains of cardiometabolic health: 1) atherosclerosis (arterial plaque accumulation), 2) dyslipidemia (unhealthy blood lipids), and 3) hyperglycemia (high blood sugar). Accordingly, three key findings emerge that comprise the three chapters of this report. 1) The quality of human sleep, specifically the degree of fragmentation, raises inflammatory-related white blood cells, thereby conferring an increased risk for atherosclerosis. This was true of sleep fragmentation assessed across a week or a single night, which predicted increasingly higher CAC scores through a mediating association with increased neutrophils. 2) Unique features of an individual’s sleep (duration, efficiency, timing, regularity) predict aspects of triglyceride metabolism under fasting (homeostatic) and post-food intake (allostatic) conditions. First, later sleep timing predicts higher fasting triglycerides, emphasizing a unique relation with triglyceride homeostasis (not allostasis). Second, this homeostatic link is mediated by increased systemic inflammation (GlycA levels). Third, and conversely, declining sleep efficiency negatively impacts post-food lipid clearance, reflecting allostatic regulation. Fourth, these effects are replicated in a second independent cohort. 3) The coupling of NREM sleep spindles and slow oscillations the night prior is associated with improved next-day peripheral glucose control. Further, this sleep-associated glucose pathway may influence glycemic status through altered insulin sensitivity, rather than through altered pancreatic beta cell function. Moreover, these associations are replicated in an independent dataset of over 1900 adults. Of therapeutic significance, the coupling between slow oscillations and spindles was the most significant sleep predictor of next-day fasting glucose, even more so than traditional sleep markers, relevant to the possibility of an EEG index of hyperglycemia. Collectively, beyond these scientific insights, these results support a sleep–cardiometabolic homeostasis framework, such that unique features of sleep act as biomarkers for unique aspects of cardiometabolic health. Importantly, these findings help reinform public health sleep-related guidelines to reduce the mortality and economic burden of cardiometabolic disease globally.

Cardiovascular disease (CVD) stands as the predominant cause of mortality on a global scale, with diabetes mellitus (DM) emerging as a prominent risk factor for CVD. Notably, adults afflicted by DM face a nearly twofold increased risk of CVD-related mortality compared to their non-DM counterparts. The present dissertation undertakes the task of formulating pre- ventive strategies for CVD within the DM patient population. The first chapter of this study endeavors to ascertain the residual risk of CVD stemming from hypertriglyceridemia (HTG) in the DM population, drawing data from the National Health Nutrition and Examination Survey (NHANES) spanning the years 2007 to 2014. Interestingly, it is observed that despite the implementation of first-line lipid-lowering therapy involving statin usage and optimal low- density lipoprotein cholesterol (LDL-C) control, approximately one-fifth of this population continues to exhibit HTG. Additionally, three-quarters of DM patients possess a moderate to high 10-year risk for CVD. The second chapter aims to assess the potential impact of two novel DM medications, namely liraglutide (glucagon-like peptide-1 receptor agonists (GLP-1 RAs)) (Chapter 2) and empagliflozin (sodium-glucose cotransporter 2 inhibitor (SGLT-2i)) (Chapter 3), on CVD prevention. The evaluation is conducted in terms of the preventive capacity for CVD events while on treatment, utilizing the NAHNES database. Followingix the meticulous application of key inclusion and exclusion criteria derived from corresponding clinical trials, an estimated 4.2 million and 1.86 million US DM patients are deemed eligi- ble for these treatments, respectively. Moreover, approximately 21,209 and 12,066 major cardiovascular events, comprising a composite outcome of cardiovascular death, myocardial infarction, and stroke, could be averted annually, respectively. The forth chapter endeav- ors to explore the impact of multifactorial risk control—encompassing body mass index, blood glucose, blood pressure, blood lipid levels, physical activity, nicotine exposure, sleep health, and dietary patterns—on the relative risk of CVD development among DM patients based on the UK Biobank database. Notably, a strikingly low percentage of only 2.2% of DM patients demonstrate ideal control over at least six of the above risk factors, and this achievement is associated with an 84% lower risk of incident CVD when compared to individ- uals without any risk factor control. The combined findings of the four chapters underscore the paramount importance of adopting an integrative approach to CVD prevention, partic- ularly in the context of DM patients. The insights gained from this research endeavor hold significant implications for shaping comprehensive strategies aimed at mitigating CVD risks and enhancing patient outcomes within this vulnerable population.

Introduction: In 2011, the National Heart, Lung, and Blood Institute recommended universal lipid screening of all youth and adolescents at ages 9-11 and 17-21 years of age. In contrast, the U.S. Preventative Services Task Force statement could not recommend screening one way or another. The difference was due to limited data on the effect of early screening and treatment, especially in the 20-35 year old population, and limited cost-effectiveness evidence for universal screening. Purpose: The purpose of this study was to examine real-world temporal trends in lipid testing, dyslipidemia, and lipid lowering therapy (LTT) utilization in ages 2-30 for 2007-2012, and to estimate the cost-effectiveness of universal vs. selective pediatric lipid screening. Methods: Temporal trends were analyzed using retrospective claims and electronic health record data from three different health care systems across the U.S. The prevalence of lipid testing, dyslipidemia, and LLT utilization were determined for each year. Two-sided Cochran-Armitage methods were used to assess trend changes. The cost-effectiveness model combined two different models. The first was a Markov Model for US 10 year-old children that predicted the distribution of the population with dyslipidemia and their percentage LDL-C reduction at age 35 under universal screening and selective screening. Data from the first model were input into the existing Cardiovascular Disease Model. The cohort was followed until age 55 and estimates of the predicted total costs and quality-adjusted life years (QALY) were calculated. Results: Comparing age groups 2-20 years and 21-30 years, the prevalence in lipid testing decreased from 4.4% to 2.0% and 9.1% to 5.7%, and the prevalence of LLT decreased from 0.04% to 0.02% and from 0.35% to 0.12% , respectively (all: p

Metabolic syndrome (MetS), a disorder with a high and growing prevalence, is a recognized risk factor for cardiovascular disease (CVD) and type 2 diabetes. It is a constellation of clinical and metabolic risk factors that include abdominal obesity, dyslipidemia, glucose intolerance, and hypertension. Unfortunately, MetS is typically underrecognized, and there is great heterogeneity in its management, which can hamper clinical decision-making and be a barrier to achieving the therapeutic goals of CVD and diabetes prevention. Although no single treatment for MetS as a whole currently exists, management should be targeted at treating the conditions contributing to it and possibly reversing the risk factors. All this justifies the need to develop recommendations that adapt existing knowledge to clinical practice in our healthcare system. In this regard, professionals from different scientific societies who are involved in the management of the different MetS components reviewed the available scientific evidence focused basically on therapeutic aspects of MetS and developed a consensus document to establish recommendations on therapeutic goals that facilitate their homogenization in clinical decision-making.

Introducción y objetivos: evaluar prospectivamente la asociación entre el consumo de huevo y el riesgo de dislipidemia en una cohorte mediterránea. Métodos: se siguieron 13.104 graduados universitarios españoles durante un periodo medio de 8 años. La dieta se evaluó al inicio utilizando un cuestionario semicuantitativo de frecuencia de consumo de alimentos repetidamente validado. Las concentraciones sanguíneas de colesterol total, lipoproteínas de alta densidad (HDL-c) y triglicéridos autorreferidas fueron evaluadas según categorías de consumo de huevo tras 6 y 8 años de seguimiento. También se evaluó la asociación entre el consumo basal de huevo y la incidencia de hipercolesterolemia, concentraciones bajas de HDL-c e hipertrigliceridemia durante el seguimiento. Resultados: se observó una asociación entre los niveles intermedios de consumo de huevo (2-4 unidades/semana frente a < 1 unidad/semana) y menor riesgo de hipertrigliceridemia con OR = 0,71 (intervalo de confianza del 95% [IC]: 0,54 a 0,93, p < 0,05) en el modelo más ajustado. Tras 8 años de seguimiento, encontramos una asociación entre un mayor consumo de huevo y menores niveles de HDL-c (p tendencia lineal = 0,02) con una diferencia ajustada de -4,01 mg/dl (-7,42 a -0,61) para > 4 vs. < 1 unidad/semana. Se encontraron menores concentraciones de triglicéridos en las tres categorías superiores de consumo de huevo en comparación con la inferior con resultados significativos para algunas de estas categorías después de 6 y 8 años de seguimiento. Conclusiones: un mayor consumo de huevo no se asoció con niveles anormales de colesterol total o triglicéridos; se encontró una asociación inversa con HDL-c como variable cuantitativa solo en uno de nuestros análisis.

Circulating lipoproteins are increasingly recognized as innate immune effector molecules. Severe hypolipidemia, which may occur with trauma or critical illness, is clinically associated with bacterial pneumonia. Importantly, pneumonia caused by Staphylococcus aureus is a major cause of morbidity and mortality in both hospital- and community-acquired pneumonia cases. Here we report that lipoprotein deficiency impairs early pulmonary innate defense against S. aureus quorum-sensing dependent pathogenesis. Specifically, apoB levels in the lung early post-infection are significantly reduced with lipoprotein deficiency, coinciding with impaired host control of S. aureus agr-signaling and increased agr-dependent morbidity and inflammation. Given that lipoproteins also inhibit LTA- and LPS-mediated inflammation, these results suggest that hypolipidemia may broadly impact post-trauma pneumonia susceptibility to both Gram positive and Gram negative pathogens. Together with previous reports demonstrating that hyperlipidemia also impairs lung innate defense, these results suggest that maintenance of normal serum lipoprotein levels is necessary for optimal host innate defense in the lung.

The overall goal of my thesis research is to further the understanding of the link among dietary trimethylamine-containing nutrients, intestinal microbiome, and cardiovascular disease (CVD) by testing the hypothesis that disease susceptibility is a transmissible trait via cecal microbial transplant. The positive correlation discovered between plasma TMAO and lesion in a panel of inbred mouse strains permitted the identification of atherosclerosis prone (high TMAO-producing, large lesion; C57BL/6J) and resistant mice (low TMAO-producing, small lesion; NZW/LacJ) to be used as donor strains for cecal microbial transplant into intestinal microbiota-suppressed recipient C57BL/6J Apoe-/- female mice. Recipients of C57BL/6J microbes on the choline diet initially had statistically significantly higher plasma TMA and TMAO as compared to choline diet NZW/LacJ recipients, and at the study endpoint, choline diet-dependent enhancement of atherosclerotic lesion area was observed in the recipients of C57BL/6J microbes but not in recipients of NZW/LacJ microbes. Examination of microbial communities from 16S rRNA bacterial gene sequencing analysis showed distinct donor communities faithfully transferred to the respective recipient mouse groups. Several donor strain-characteristic taxa were identified in recipient groups whose proportional abundances correlated with plasma TMAO and/or lesion area. The second set of studies explores thrombosis potential and platelet reactivity, the proximal events in heart attack and stroke, for a relationship with the microbiome. Plasma TMAO is associated with a 2-fold increased risk for thrombotic events in a large cohort (n=4007). TMAO enhances agonist dependent platelet responsiveness by increasing the release of intracellular calcium stores. In mice, provision of TMAO or dietary choline (solely in the setting of an intact microbial community) enhances platelet aggregation ex vivo and shortens the time to cessation of blood flow in vivo. Cecal microbial transplant study showed that germ-free recipients of the disease-prone donor microbes were transmitted bacterial communities, choline diet-dependent enhanced TMA lyase activity, elevated plasma TMAO, and prothrombotic phenotypes as compared to recipients of disease-resistant donors. Collectively, the results of these studies suggest that the body and its microbial inhabitants interact to promote health or disease. Targeting microbial communities could be an adjunct treatment for the management of cardiometabolic disease in the future.

Background: Familial Hypercholesterolemia (FH) is estimated to affect one in every five hundred people. FH is a lifelong condition that if untreated can lead to premature coronary heart disease (CHD) and possibly death. In spite of the high prevalence, internationally FH is both underdiagnosed and undertreated, leaving a large population of people at a greatly increased risk for premature CHD. There are effective treatments available that can significantly improve long term survival. Methods: The prevalence of FH at CCHMC for individuals between age 2 and 21 was calculated using i2b2, a research data warehouse at CCHMC. A chart review was completed on 180 patients who were seen between 1/1/2011 and 7/15/2013 at CCHMC and met the inclusion criteria. A 42 item survey was developed to evaluate parental knowledge, attitudes and interest in genetic counseling and genetic testing. 171 surveys were sent out, 86 included an educational brochure created by the FH Foundation and 85 did not include the brochure. Results: The prevalence of FH in the selected population was 0.04%. 1% of individuals between the ages of 9 and 11 received cholesterol screening at CCHMC. Of the 180 individuals included in the chart review, 146 (81%) had seen a lipid specialist, 47 (26%) had a diagnosis of FH, 105 (58%) had a diagnosis of dyslipidemia or hyperlipidemia, 28 (16%) had been prescribed a statin, 0 (0%) had discussed FH with a genetic counselor. Of the 171 surveys sent, 76 surveys were completed with a response rate of 44%. Respondents whose child had been prescribed a statin had a higher mean knowledge score than respondents who children had not been prescribed a statin (p = 0.0267). If the respondent reported that a healthcare provider had talked to them about FH there was a trend towards significant associated with knowledge score (p = 0.0606). Receiving the educational brochure, having a diagnosis of FH, reported use of the internet to look up FH, annual house hold income and a reported family history of hypercholesterolemia were not associated with increased knowledge. Conclusions: Results from this study supports existing data regarding the under diagnosis of FH and the lack of adherence to routine cholesterol screening. Participants who reported a health care provider had previously spoken to them about FH were more knowledgeable compared to those who had not. Given the important role healthcare providers play in patient education, incorporating genetic counselors into the counseling and risk assessment of these patients and families may increase overall disease knowledge as well as adherence to cholesterol screening and genetic testing guidelines.

The Effect of Hypovitaminosis D on Risk for Cardiovascular DiseasePatricia G. Weyland, MS, RN, FNP-BC, CDE, PhD (c)AbstractBackground: Serum 25-hydroxyvitamin D (25(OH)D) levels have been found to be inversely associated with both prevalent and incident cardiovascular disease (CVD) risk factors; dyslipidemia, hypertension and diabetes mellitus. Objective: This review looks for evidence of a causal association between low 25(OH)D levels and increased CVD risk. Method: We evaluated journal articles in light of Hill's criteria for causality in a biological system. Results: The results of our assessment are as follows. Strength of association: many randomized controlled trials (RCTs), prospective and cross-sectional studies found statistically significant inverse associations between 25(OH)D levels and CVD risk factors. Consistency of observed association: most studies found statistically significant inverse associations between 25(OH)D levels and CVD risk factors in various populations, locations and circumstances. Temporality of association: many RCTs and prospective studies found statistically significant inverse associations between 25(OH)D levels and CVD risk factors. Biological gradient (dose-response curve): most studies assessing 25(OH)D levels and CVD risk found an inverse association exhibiting a linear biological gradient. Plausibility of biology: several plausible cellular-level causative mechanisms and biological pathways may lead from a low 25(OH)D level to increased risk for CVD with mediators, such as dyslipidemia, hypertension and diabetes mellitus. Experimental evidence: some well-designed RCTs found increased CVD risk factors with decreasing 25(OH)D levels. Analogy: the association between serum 25(OH)D levels and CVD risk is analogous to that between 25(OH)D levels and the risk of overall cancer, periodontal disease, multiple sclerosis and breast cancer. Conclusion: All relevant Hill criteria for a causal association in a biological system are satisfied to indicate a low 25(OH)D level as a CVD risk factor.

Controlling blood lipids is a major public health challenge of our time. The pleiotropic hormone-like polypeptide fibroblast growth factor 21 (FGF21) was recently recognized as a potent modulator of lipid and glucose metabolism and a promising treatment strategy for obesity related metabolic disorders, including dyslipidemia. A cost effective and practical alternative to the administration of recombinant FGF21 is to stimulate endogenous FGF21 production through diet. Our research identified (R)-a-lipoic acid (LA), a naturally occurring enzyme cofactor and dietary molecule found in green leafy vegetable and red meat, as an inducer of FGF21 expression. LA stimulated FGF21 production, demonstrated by a 3-fold increase in circulating FGF21, in the obese animal by upregulating hepatic Fgf21 expression. Concomitantly, feeding LA to dyslipidemic obese rats corrected high blood triacylglycerol levels and lowered abdominal adiposity by stimulating liver gene expression of peroxisome proliferator-activated receptor a (PPARa) target genes involved in lipid disposal and conjointly decreasing de novo lipid synthesis as a two-sided complementary mechanism to mitigate lipid burden. Since the metabolic effects of LA mimic those of FGF21, we posit that FGF21 mediates the beneficial phenotype evoked by LA and sought to determine how LA and its reduced form, (R)-a-dihydrolipoic acid (DHLA), induce the Fgf21 promoter. To that end, we employed HepG2 hepatocellular carcinoma cells under fed and starved states and in the presence of LA and DHLA. Serum withdrawal markedly induced FGF21 mRNA abundance from 24 to 48 hours after serum removal. Both LA and DHLA induced FGF21 mRNA levels at 36 hours after serum removal when compared to vehicle control. To further determine the regulatory mechanism of FGF21 transcription, we have begun to develop the FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) assay coupled to qPCR to isolate and identify active regulatory promoter regions. We designed and validated a collection of primers specific for human FGF21 promoter that will allow the functional mapping of a 4,573-bp 5’ UTR region. Advisor: Regis Moreau

Abstract: Introduction: Trans 11-18:1 (vaccenic acid, VA) is the predominant ruminant-derived trans fatty acid (TFA) in the food chain and is also the precursor to endogenous synthesis of cis-9, trans-11 conjugated linoleic acid (CLA). Literature documents numerous health benefits associated with CLA, which is a minor component of natural TFA. A few recent studies have reported hypolipidemic properties of VA. However, independent bioactive properties of VA during dyslipidemia and insulin resistance remain elusive. Objectives: To determine whether VA can enhance the putative hypolipidemic effects of CLA. To investigate whether enriching dairy fat with VA can favourably modulate whole body energy use/storage. Methods and Results: Hypolipidemic effects of dietary supplementation with CLA on dyslipidemia were enhanced by the addition of VA in a rat model of metabolic syndrome (the JCR:LA-cp rat). VA was also found to reduce total body fat (-6%) while stimulating adipose tissue re-distribution (reduced mesenteric fat (-17%) while increasing inguinal fat mass (29%)). VA fortification increased metabolic rate concomitantly with an increased preference for whole body glucose utilization for oxidation and increased insulin sensitivity (lower HOMA-IR index (-59%)). Additionally, VA re-equilibrated intestinal and hepatic lipid homeostasis (reduced TG secretion) while exerting differential transcriptional regulation in both organs. Targeted lipidomic analyses revealed that VA can regulate concentrations of endocannabinoids in a tissue-specific manner. Conclusions: VA has hypolipidemic effects independent of CLA and can favorably modulate whole body energy metabolism and lipid homeostasis when added to a dairy-derived diet. The regulatory effect of VA on the endocannabinoid system may have potential therapeutic applications. Based on these results, enriching dairy products with VA (either naturally or by fortification) may maximize the health value of dairy-derived fats. Findings also support the need for nutrition guidelines focused on eliminating industrial forms of TFA from processed foods, as opposed to all TFA per se.

Abstract: Liver derived low-density lipoprotein and intestinal derived chylomicron remnant are contributors to dyslipidemia and cardiovascular disease (CVD). In clinical practice, the most common medication to reduce fasting LDL-cholesterol is statins. However, for many individuals prescribed statins, a substantial ‘residual risk’ of CVD remains. Therefore, effective nutraceutical approaches that have multiple targets to lower cholesterol secreted by both the liver and the intestine remain attractive. The objective was to assess the lipid lowering efficacy of novel formulations: chitin-glucan, phytosterol, the red yeast rice, using a rodent model of dyslipidemia. It was observed that these active ingredients all independently reduced fasting plasma lipids. Further, the combination of these ingredients, with 1mg/kg red yeast rice, was the most effective at reducing both fasting and non-fasting plasma lipids. The findings provide pre-clinical support for the combined use of these ingredients as a nutraceutical alternative to classic lipid lowering pharmacotherapies.

ABSTRACT SANDRA C. METCALF Associations of Youth Weight Status Categories and Cholesterol Levels: Analysis of Data from the National Health and Nutrition Examination Survey (NHANES) (Under the direction of RODNEY LYN, PhD, Assistant Professor) Obesity is recognized as a risk factor for dyslipidemia, however studies specific to overweight youth and dyslipidemia are scarce. Nonetheless, expert bodies have established BMI at the 85th percentile or greater as the threshold for “at risk” in youth and advocate for lipid screening. This study analyzed associations of weight categories and occurrence of dyslipidemia among a multi-racial sample of youth 6 to 18 years of age. In this NHANES cohort, overweight youth were at significant increased risk of dyslipidemias of total cholesterol, high-density lipoprotein cholesterol and non-high-density lipoprotein cholesterol. Obese children and adolescents demonstrated even higher risk. This study supports the hypothesis that overweight youth are at increased risk for dyslipidemia. Despite the increased risk, test agreement for overweight and dyslipidemia was poor. BMI at the 85th percentile did not provide good discrimination in detecting children with dyslipidemia and use of this threshold warrants further review.

Gene-environment interactions are increasingly understood to be a primary contributing factor in the etiology of cardiovascular disease. Environmental factors include person and societal-level variables. Genome factors include sequence variation and mechanisms controlling genome expression. These regulatory mechanisms are the nexus of gene-environment interactions, and can be transient or adaptive; the latter are termed epigenetics. Some genetic characteristics are highly homogeneous within ethnic groups, due to a combination of geographically derived natural selection and environmental factors. South Asians are a population with disproportionately high cardiovascular disease burden. A prevalence study of cardiovascular risk factors in this population provides evidence that common risk factors are alterations in cholesterol metabolism; specifically low high-density lipoprotein cholesterol with elevated triglycerides. These initial observations led to a second study yielding evidence that abdominal adiposity is not a necessary precursor to the development of this dyslipidemia, challenging previous assumptions. A third study found pro-atherogenic risk profiles in South Asians are present decades earlier than in other populations. While these risk factors have genetic underpinnings, the added effects of a changing environment (i.e., Westernization of South Asians) are not known. MicroRNA are an epigenetic post-transcriptional regulatory mechanism of messenger RNA translation, and are potential biomarkers of pathophysiology and response to interventions. MicroRNA in human blood are differentially expressed in numerous disease conditions, and in vitro studies indicate that microRNA play a role in regulation of cholesterol metabolism in response to the extracellular environment. Methods to quantify microRNA include microarray, in which a large number of microRNA targets are screened. Microarray results are then validated with the more sensitive quantitative polymerase chain reaction method. Using microRNA arrays and a case-control design of pooled bio-specimens, a fourth study identified 16 candidate microRNA biomarkers of this atherogenic profile, which were then verified by quantitative polymerase chain reaction. Several of these are known to target messenger RNA involved in cholesterol metabolism. These results are promising evidence for translational application of microRNA as clinical biomarkers elucidating underlying genetic determinants of atherogenic risk in South Asians. Further research is needed to determine whether microRNA expression in blood is sensitive to cardiovascular risk reduction interventions.

ATP-binding cassette transporters ABCA1 and ABCG1 initiate reverse cholesterol transport generating HDL particles, whereas ABCG5/G8 promote biliary cholesterol secretion thereby facilitating the last step of reverse cholesterol transport. Mutations in the leptin axis result in obesity and dyslipidemia in ob/ob and db/db mice. These mice have defective HDL clearance, increased plasma cholesterol and decreased biliary cholesterol elimination. My studies demonstrate that ABCG5/G8 protein is low in these animals and can be restored with caloric restriction or leptin replacement. To directly test whether ABCG5/G8 alone is able to correct reverse cholesterol transport defect, liver specific ABCG5/G8 expression was achieved in db/db mice by administration of adenoviral ABCG5 and ABCG8. Restoration of biliary cholesterol is able partially to correct dyslipidemia in obese mice, but only in the presence of ezetimibe, an inhibitor of cholesterol absorption. ABCG5/G8 is the body’s primary defense against toxic effects of plant sterols. Plant sterols are used as cholesterol lowering food supplements. However, increased plasma plant sterol concentrations are associated with vascular lesions in dyslipidemic patients and animals. My in vitro studies demonstrate that individual plant sterol alter ABCA1 and ABCG1 abundance, cholesterol efflux and inflammatory cytokine secretion in macrophage foam cells at levels found in humans that consume plant sterol supplements.

Abstract: Trans-11 vaccenic acid (VA) is the predominant trans fatty acid in dairy fat and is the major precursor to endogenous synthesis of cis9,trans11-conjugated linoleic acid (CLA) in humans and animals. Epidemiological studies have shown the positive association between trans fat intake and incidence of coronary heart disease. Nevertheless, CLA, categorized as a group of trans fatty acids, has been shown to possess anti-carcinogenic, hypolipidemic and anti-diabetic benefits in several animal models as well as certain human populations, possibly via activating peroxisome proliferator-activated receptor (PPAR) related metabolic pathways. The subsequent effort in enriching CLA in dairy products (e.g. butter) has led to a concomitant increase in VA, whose bioactivity and health implications were not fully appreciated. Interestingly, VA is the major natural trans fat found in the diet. Therefore, the objectives of this thesis were to assess the effect of dietary supplementation of synthetic VA on lipid metabolism especially during conditions of dyslipidemia and metabolic syndrome, and to delineate the intestinal and hepatic metabolic pathways potentially modulated by VA. The JCR:LA-cp rat model, when homozygous for the cp trait (cp/cp), develop leptin receptor deficiency which leads to symptoms of metabolic syndrome and pre-diabetes including obesity, insulin resistance, hepatic steatosis, hypertriglyceridemia and exacerbated production of hepatic very low-density lipoproteins and intestinal chylomicrons (CM). Gas chromatography analysis on nascent lymph shows that VA was effectively absorbed into the intestine. In addition, VA from natural source (i.e. beef fat) showed higher intestinal bioavailability compared to synthetic VA. Dietary supplementation of 1.0% (w/w) synthetic VA to JCR:LA-cp rats (but not lean healthy controls) demonstrated a profound reduction in plasma triglyceride, total cholesterol, low-density lipoprotein-cholesterol, non-esterified fatty acid and haptoglobin concentrations (51%, p

Obesity and related metabolic disorders have reached epidemic levels with dire public health consequences. Efforts to stem the tide focus on behavioral and pharmacological interventions. Several hypolipidemic pharmaceutical agents target endogenous lipid receptors, including the peroxisomal proliferator activated receptor alpha (PPAR alpha) and cannabinoid receptor 1 (CB1). To further the understanding of these clinically relevant receptors, we elucidated the biochemical basis of PPAR alpha activation by lipoprotein lipolysis products and the metabolic and transcriptional responses to elevated endocannabinoid signaling. PPAR alpha is activated by fatty acids and their derivatives in vitro. While several specific pathways have been implicated in the generation of PPAR alpha ligands, we focused on lipoprotein lipase mediated lipolysis of triglyceride rich lipoproteins. Fatty acids activated PPAR alpha similarly to VLDL lipolytic products. Unbound fatty acid concentration determined the extent of PPAR alpha activation. Lipolysis of VLDL, but not physiological unbound fatty acid concentrations, created the fatty acid uptake necessary to stimulate PPAR alpha. Consistent with a role for vascular lipases in the activation of PPAR alpha, administration of a lipase inhibitor (p-407) prevented PPAR alpha dependent induction of target genes in fasted mice. Apolipoprotein CIII, an endogenous inhibitor of lipoprotein lipase, regulated access to the lipoprotein pool of PPAR alpha ligands. Our results support a role for the local generation of PPAR alpha ligands by lipase activity. The endocannabinoid system regulates diverse physiological functions, including energy balance. While loss of CB1 signaling has more pronounced effects on lipid parameters than expected based on weight loss alone, direct demonstration of endocannabinoid regulation of lipid metabolism is lacking. To test the effects of endogenously produced cannabinoids on lipid metabolism, independent of alterations in food intake, we analyzed tissues from mice treated with IDFP, an organophosphorus inhibitor of endocannabinoid breakdown. IDFP administration inhibited hepatic monoacylglycerol lipase leading to elevated levels of 2-arachidonylglycerol. We found that IDFP administration caused accumulation of apoE depleted VLDL. HDL particles accumulated apoE and failed to transfer it to VLDL in vitro. Importantly, these effects were prevented by pharmacological inhibition of CB1 and absent in plasma from CB1 mice. IDFP also caused a CB1-dependent increase in hepatic triglycerides. Thus, endocannabinoids inhibit the transfer of apoE from HDL to VLDL leading to apoE depletion of triglyceride rich lipoproteins. Microarray analysis allowed us to determine the effects of IDFP on expression of genes involved in lipid metabolism and to discover novel cannabinoid responsive genes. IDFP increased expression of lipogenic and SREBP2 target genes in a CB1-dependent fashion. On a global scale, pre-administration of a CB1 antagonist prevented many of the IDFP induced alterations in gene expression. IDFP decreased expression of genes involved in amino acid metabolism and inflammation. PCR analysis of selected mRNAs confirmed several of the key array findings. Our work indicates that endocannabinoids exert a large influence on hepatic lipid metabolism independent of food intake and suggest that peripherally restricted CB1 antagonists may be of therapeutic value. Overall, these findings shed light on the endogenous mechanisms of PPAR alpha activation and the hepatic responses to Cb1 activation. This information may help guide the continuing effort to develop treatments for metabolic disease.

Metabolic syndrome is a cluster of disorders including central obesity, hypertension, dyslipidemia, and hyperglycemia. Today's metabolic syndrome definitions identify central obesity by waist circumference (WC) measurements. A recent pilot study suggests that cut-points derived from a waist-to-height ratio (WHtR), or Index of Central Obesity (ICO), is a more accurate measurement of central obesity. This study compared the association between the metabolic syndrome components and central obese parameters (ICO and WC) among the white, black, and Hispanic adults in the United States. The subjects' data was obtained from the 2005-2006 National Health and Nutrition Examination Survey. ICO was highly correlated with metabolic syndrome components among white subjects and the least correlated in Hispanic subjects. Multivariate logistic regression analysis did not indicate that ICO was a better parameter for metabolic syndrome than WC. Other WHtR cut-points may be more sensitive in predicting metabolic syndrome components than the values used in this study.