NC1199: N-3 polyunsaturated fatty acids and human health and disease

(Multistate Research Project)

Status: Active

NC1199: N-3 polyunsaturated fatty acids and human health and disease

Duration: 10/01/2012 to 09/30/2017

Administrative Advisor(s):


NIFA Reps:


Statement of Issues and Justification

The need as indicated by the stakeholders: N-3 fatty acids are widely recognized as beneficial dietary components. In support, numerous scientific societies or governing bodies such
as the American Heart Association, American Diabetes Association, American Dietetic Association, Institute of Medicine (IOM) Report on Seafood Choices and the FAO/WHO Expert
Consultation on the Risks and Benefits of Fish consumption recommend consuming oily fish at least twice per week. Although the IOM of the National Academy of Sciences has
established Dietary Reference Intakes (DRIs) for ±-linolenic acid, they do not provide DRIs for longer chain n-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic
acid (DHA). The IOM highlights the need for addition research to look at the roles of n-3 fatty acids in numerous diseases including cancer, inflammatory and metabolic disorders
and degenerative diseases of the central nervous system. The problem: Although numerous scientific bodies recommend n-3 fatty acid consumption, the effects of n-3 fatty acids on
health outcomes is poorly characterized for many diseases. Additionally, the mechanism through which n-3 fatty acids work to elicit the beneficial effects is also only partially known.
As evidenced by the lack of recommendations by the IOM for EPA and DHA, the specific effects of different n-3 fatty acids requires further investigation before accurate DRIs can be
developed. An underlying problem regarding n-3 fatty acids is the confusion amongst the public about the health benefits of short chain and long chain n-3 PUFA fatty acids. Members of the NC-1039 Committee have been at the forefront of n-3 fatty acid research. This group brings together a wide-range of expertise that uniquely can expedite scientific
discovery and translation. The overall goal of this application is to further characterize the functional effects of n-3 fatty acids on health-related outcomes, the mechanisms through
which they exert their effects, and to provide scientific-based information via an eXtension website to the public domain.


This project addresses the goals of the Experimental Station Committee on Organization and Policy Science Roadmap Challenge 5 - We must improve human health, nutrition, and
wellness of the US population. The current proposal addresses this challenge directly by focusing on the role of dietary n-3 fatty acids on numerous health conditions and
translating information on n-3 fatty acids to the public. This project also addresses two Food and Nutrition Crosscutting Research Areas (NCRA) and Objectives of the North Central
Regional Association. The two crosscutting objectives to be addressed by NC-1039 are: Research Objective 1 - Emphasize research that expands our understanding of the
relationship between diet, health and disease prevention with particular focus on antioxidants, dietary lipids, functional foods/nutraceuticals, nutrient bioavailability, nutrient
regulation of gene expression and nutrition and physical activity, and Research Objective 2 - Design effective nutrition education programs and delivery methods that modify human
behavior such that individuals including those most at risk (pregnant women, infants, adolescents and the elderly) choose healthier diets. Aims 1 and 2 of this project will address
NCRA objective 1 and 2 and Aim 3 will address NCRA objective 2.


In addition to serving as a source of energy, n-3 fatty acids have numerous functions in the body and contribute to overall health by targeting multiple biological systems. Moreover,
n-3 fatty acids are potent signaling molecules that influence biology beyond their basic nutritional value. Because of these broad ranging effects of n-3 fatty acids, the complexity of
the mechanisms through which n-3 fatty acids work to influence health have not been fully elucidated. Although the effects of long chain n-3 fatty acids on a few health conditions
such as cardiovascular disease (CVD) and hypertriglyceridemia and the effect of the n-3 DHA for optimal infant growth and cognitive development have been well-documented, their
effects on etiology or outcomes of other conditions have not been comprehensively examined. Because of this lack of knowledge, their remains inconsistencies in dietary
recommendations for the type and source of n-3 fatty acids and, as a result, confusion amongst the public. Thus, there is a clear need to better define and communicate the benefits
of n-3 fatty acids. A groups such as NC-1039 whose recommendations and messages are based on the most current science must emerge as the trusted source of n-3 health
information to combat the misinformation being provided to the public by non-scientific, non-medical organizations.


Importance of the work: The lack of a specific DRI for an important nutrient such as long chain n-3 fatty acids (EPA and DHA) results directly from insufficient data to support a DRI.
This is concerning given the well-documented effects of long chain omega-3 fatty acids on lowering the risk for cardiovascular disease, the number one leading cause of mortality in
the United States. Despite the lack of a specific DRI for the long chain n-3 fatty acids, consumption of EPA and DHA is recommended by most health experts. However, the
relationship between n-3 fatty acids and health disorders outside of CVD are poorly understood. Studies have implicated a beneficial role of n-3 fatty acids promotion of infant
cognitive development and prevention of numerous diseases including cancer, bipolar disease, hypertension, Type 2 Diabetes, obesity, non-alcoholic fatty liver disease,
inflammatory/autoimmune diseases (i.e. asthma, rheumatoid arthritis, inflammatory bowel disease), cognitive development, reproduction, Alzheimers and other dementia-related
diseases, lupus, osteoporosis and depression. Most studies examining n-3 fatty acids are retrospective and even fewer studies delve into the mechanism through which n-3 fatty
acids provide preventative or therapeutic effects. Therefore, a clear need exists for more research on the health effects of n-3 fatty acids. The first two aims of this project will
specifically address the effects of n-3 fatty acids on different disease outcomes and the mechanism through which n-3 fatty acids elicit their health-promoting effects.


Despite the general recommendation for Americans to consume n-3 fatty acids, actual intakes remain low. US intakes of n-3 fatty acids is ~1.6 g/d, of which 1.4 g is ALA and 0.1-
0.2 g is EPA and DHA (1). These intake values are well below those recommend by AHA and other governing bodies that range from 0.3 to 0.9 g/d. Based on these data, it is clear
that there is a need to better communicate the benefits of EPA and DHA to the general public in an attempt to increase dietary consumption. Because of the lack of data, there is also
confusion amongst the public regarding the role of n-3 fatty acids in preventing or treating specific diseases. Taken together, a readily available and accurate source of information
on health benefits of n-3 fatty acids for the general public is needed. The third aim of this project involves the launch and maintenance of a website that will specifically address
these needs.


Consequences if not done: Without this research, the advancement of our understanding of n-3 fatty acids in the prevention and treatment of diseases will be slowed. This is
important because of the prevalence of these diseases in the American population and their serious impact on the healthcare economy. It is imperative that we not lump all n-3 fatty
acids together with the assumption that they have similar biological properties. The USDA needs a unified, evidence based, consortium of fatty acid nutrition experts such as
members from the NC-1039 to present accurate information to the public. To this end, the n-3 fatty acid site of the eXtension website was developed to provide answers to
questions commonly asked by the public about n-3 fatty acids and to act as an educational resource for medical and public health professionals. The most effective methods to
implement dietary behavior change is rigorous experimentation, particularly in relation to reducing total fat intake. However, these educational theories have not been applied to the
area of n-3 fatty acid intake and without this data success is questionable. In addition, education to increase intake of n-3 fatty acids is further complicated because it contradicts
previous nutrition communications recommending dietary fat reduction. Therefore, without these studies, n-3 PUFA as a tool for optimum growth and development, health
promotion and disease prevention will remain unclear to the biomedical community.


Technical feasibility of the research: The scientists that make up the technical committee on NC-1039 are leaders in their respective disciplines as it relates to the n-3 fatty acid
research. The methods outlined in this proposal are standard procedures developed and perfected by each of the investigators. As a result, each member provides a unique
perspective to the problem, when seamlessly blended, will result in definitive answers. Each investigator has an established track record of publications validating his/her own
expertise in performing the relevant methodologies need to accomplish our objectives. This applies to the biological as well as the social science portions of the proposal.


Advantages for doing the work as a multistate effort: The NC-1039 Research Project addresses the role of dietary n-3 fatty acids in the promotion of health. No single station has the
expertise or resources to investigate all the components needed to establish health driven guidelines for n-3 fatty acid intakes. We will put forth an integrative effort that combines
the strengths and capacities of each investigator. In the case of the basic scientists, numerous collaborations are outlined in this project that bring together the expertise of multiple
individuals to perform studies that otherwise could not have been done alone. Similarly, the eXtension site will rely upon the broad and extensive expertise of this group to provide
web content that maintains the site and makes it an effective education tool.


Significance and impact from successful completion of the proposed work: Upon completion, this work will result in numerous advances in public health. First, we expect data
generated from the aims outlined herein to expand our knowledge on the effects of n-3 fatty acids on disease outcomes, thereby, coming closer to confirming specific health benefits
of n-3 fatty acids. Ultimately, we anticipate that these data will also lead to specific RDIs for individual n-3 fatty acids. Secondly, we expect to further understand the mechanisms of
how omega-3 fatty acids effect different biological systems, which will both advance our understanding of n-3 fatty acids and provide insights into the development of specific
diseases. Third, the launch and maintenance of the n-3 eXtension website will provide an easily accessible and accurate avenue for translating our knowledge of health effects of n-3
fatty acids to the public. This website has the potential to increase n-3 fatty acid consumption and to point people towards the use of n-3 fatty acids as a preventative or therapeutic
agent against specific diseases. Taken as a whole, we expect the NC-1039 to provide a significant and lasting effect on improving health through the consumption of n-3 fatty acids.

Related, Current and Previous Work

Members of the NC-1039 searched the literature in formulating this section of the proposal. The CRIS database was searched using the terms omega-3 polyunsaturated fatty acids or n-3 polyunsaturated fatty acids. Twenty-seven projects were identified and, of these, nine were NC-1039 investigators.
The overall goal of the previous NC1039 project (2007-2012) was to provide a database to establish the n-3 fatty acid forms and amounts that meet dietary guidelines for optimal health and disease prevention. This goal was developed in part due to the lack of DRIs by the National Academy of Sciences and Institute of Medicine for specific long chain n-3 fatty acids. The justification for absence of a DRI for EPA and DHA was that insufficient data was available to support a recommendation (1, 2).
The following objectives were proposed for NC1039 for the 2007-2012 period to address our overall goal:
Objective #1: To determine the health promotion and disease prevention effects of both the forms and the amounts of n-3 fatty acids by correlation with tissue functions, and with alterations in biomarkers, relevant to optimal health and disease prevention.
Objective #2: To examine dietary levels of both form and amount of n-3 fatty acids that promote health and reduce disease that are relevant and achievable in human diets using the human equivalent dose method (allometric scaling) in rodent models.
Objective #3: To develop, test and disseminate effective means for translating research on the health promoting and disease preventing effects of n-3 fatty acids into consumer food choices.
We have made significant progress in meeting these objectives and the following sections highlight our progress and review the significance of these findings to the area of n-3 fatty acid form and amount in human health and disease.
Objective #1: To determine the health promotion and disease prevention effects of both the forms and the amounts of n-3 fatty acids by correlation with tissue functions, and with alterations in biomarkers, relevant to optimal health and disease prevention.
The role of specific fatty acids, including n-3 fatty acids, on central regulation of energy metabolism is not known. MN Station performed studies testing the impact of centrally delivered fatty acids on food intake and energy metabolism have revealed a unique role for n-3 fatty acids. Specifically, infusion of DHA into the 3rd ventricle (targeting the hypothalamus) robustly decreased food intake and altered hypothalamic neuropeptide expression whereas infusion of palmitate, a saturated fatty acid, had no effects on the same parameters (3). These data suggest that part of the effects of n-3 fatty on energy metabolism and perhaps other biological processes may be due to their direct effects on hypothalamic signaling. This is the first study to show that DHA alters hypothalamic signaling and opens novel research avenues for further investigation of n-3 fatty acids as important signaling molecules controlling energy metabolism via the central nervous system.
The Colorado Station performed studies examining inflammatory markers in relation to DHA intake and correlated with markers of nutritional status (serum albumin) and renal status in feline patients in stage 2 and 3 chronic kidney disease. The results indicate that DHA status may decrease inflammation in chronic kidney disease and that high DHA intakes may protect against malnutrition-inflammation syndrome as indicated by higher body condition scores and serum albumin in felines patients with chronic kidney disease.

Studies at the Nebraska Station have demonstrated that the regulatory and metabolic impact of dietary long chain polyunsaturated fatty acids, AA, EPA and DHA, differ from that of the 18 carbon polyunsaturated fatty acids, linoleic and linolenic acids. From these studies we determined the metabolic profile of blood lipids and transcription profile of liver is more similar when mice are fed diets containing saturated fat or polyunsaturated fats derived from plant sources then when mice are fed diets enriched in the marine-based n-3 fatty acids. Importantly, serum and hepatic triglycerides and cholesterol were 3-fold and 50% higher respectively when mice were fed the saturated and plant polyunsaturated diets compared to a mixture of DHA and AA (4). Transcription profiling of livers from the same animals identified 80 genes as differentially expressed between diets. Of these 80, expression of 57 were significantly different between the saturated and plant-based polyunsaturated diets, while only seven were found to be in common between plant-derived polyunsaturated fatty acids and marine n-3 fatty acids (5). Thus, these studies identify important differences between the short and long chain polyunsaturated fatty acids and provide mechanistic insights into the genes and pathways responsible for the divergent effects of the different fatty acids.

Studies showing that the n-3 fatty acids SDA and EPA have anti-inflammatory effects on primary mouse adipose tissue stem cells by reducing production of interleukin-6 in response to inflammatory stimuli. The North Dakota Station developed a new immunohistochemical method to detect how toll-like receptor-2 and transcription factor nuclear factor-kappa mediate inhibitory effects of SDA and EPA. Development of these particular methods enabled us to determine target proteins and exact intracellular signaling pathways underlying anti-inflammatory effects of n-3 fatty acids in adipose stem cells. We also showed that dietary EPA feeding prevents and reverses insulin resistance in high-fat diet-induced obese C57BL/6J mice via modulation of adipose tissue inflammation, which may be mediated by increasing plasma adiponectin concentrations. We also showed that EPA supplementation of HF diets reduce inflammation, lipogenesis and fatty acid oxidation. As a whole, the studies provide novel data for the role of n-3 fatty acids as anti-inflammatory agents involved the etiology of Type 2 daibetes.

Studies at the North Carolina Station have focused on studying the impact of high doses of n-3 fatty acids on the function of B cells. Specifically, we have identified that a mixed fish/flaxseed oil diet enhances B cell activation, as measured by upregulation of B cell markers and secretion of cytokines (6). We are currently testing the impact of physiological doses of fish oil (~4 g/day) on B cell activation. Mechanistically, we are studying how n-3 fatty acids, relative to controls, impact the biophysical and biochemical organization of lipid raft domains. We have used a combination of advanced imaging (FRET, TIRF) and biochemical methods (NMR, detergent extraction) to show that n-3 fatty acids disrupt the clustering of lipid rafts (7). The effects are driven by changes in the lateral organization of cholesterol and direct incorporation of DHA into rafts. These findings will impact our understanding the biological mechanisms through which n-3 fatty acids affect cell signaling and related metabolic processes.
Objective #2 To examine dietary levels of both form and amount of n-3 PUFA that promote health and reduce disease that are relevant and achievable in human diets using the human equivalent dose method (allometric scaling) in rodent models.
Currently there are no guidelines providing assistance as to how to formulate human equivalent diets for rodents to improve translation of data from experimental research models to humans. The Tennessee Station established a mathematical model for allometric scaling of the levels of dietary n-3 and n-6 PUFA in a rodent model that would mimic a human equivalent dose using the US DRIs (Dietary Reference Intakes) as the human endpoint. The overall intent of this research was to test our theoretical model for allometric scaling for interspecies extrapolation. Following adjustment and validation of the mathematical model, a series of experiments were performed in C57BL/J6 mice comparing dietary alpha-linolenic acid (ALA), and eicosapentaenoic acid (EPA) at three supplemental levels each on putative tissue human biomarkers in blood (plasma and erythrocyte phospholipids)(8). These results were qualitatively compared to archival human data. The data suggests that comparable levels of n-3 and n-6 fatty acids in diets between species can be made based on metabolic differences (differences in caloric consumption to maintain body weight). This mathematical model using a surrogate of metabolic activity instead of differences in body weight for allometric scaling would better equate interspecies translation and accommodate the differences in metabolic disparity between rodents and humans. This potentially improves pre-clinical screening value for experimental animal models to predict equivalent human biological responses.

Employing the allometric scaling approach, the Wyoming Station incorporated n-3 fatty acids into a rodent diets at a percent of calories equivalent to human dietary n-3 intakes as fish oil (EPA/DHA), EPA and DHA alone, SDA and ALA to examine ovulation in a rat model. Using this approach, it appears that there is a differential effect in the ovaries that does not follow the typical cardiovascular/hypotriglyceridemic effect displayed by n-3 fatty acids. ALA at mild elevations was as effective as EPA/DHA at enhancing ovulation while at low levels demonstrated the same non-effect as low EPA/DHA. SDA was more effective at promoting ovulation over EPA alone. Alterations in ovulation appear to be attributable to two factors; an increase in constitutive cyclooxygenase-1 expression induced by the n-3 fatty acids and elevated 3-series prostaglandin biosynthesis in response to diets containing DHA. Research is ongoing in women and still under analysis as to if the effect found in rats may be of benefit in polycystic ovary syndrome. At this juncture, EPA/DHA may lead to a beneficial reduction in estrogen and an elevation in sex hormone binding globulin, possibly reducing the risk of estrogen dependent disorders and cancers.
Objective #3 To develop, test and disseminate effective means for translating research on the health promoting and disease preventing effects of n-3 PUFA into consumer food choices.
In order to effectively communicate consumer-driven messages regarding the health benefits of omega-3s and their consumption using social media distribution venues, formative research, peer-reviewed message development, and testing has been performed. The decision to use social media platforms was based on previous research performed by the New Jersey, Colorado, Nebraska and Kansas Stations that suggested these means of communications were preferred by both consumers and health professionals.

Following formative research that identified omega-3 rich foods currently available in the marketplace, a podcast (that can be used on any mp3 player) was developed to inform shoppers about the health benefits of omega-3s and how to increase the consumption of n-3 fatty acid rich foods. The podcast was designed as a grocery store tour to guide consumers to find n-3 fatty acid rich foods in each aisle of the store, while providing health information designed to increase consumers intentions to purchase n-3 fatty acid rich foods. Use of the podcast while shopping showed consumers perceived abilities to shop for n-3 fatty acid rich foods, perceived importance regarding buying n-3 rich foods, increased purchase of n-3 fatty acid rich foods, and perceived intent to purchase n-3 rich foods increase as a result of podcast use. The podcast has been disseminated through Rutgers at iTunes (title: A Grocery Store Tour for n-3 Rich Foods), where it is a free download accessed through iTunes. It will also soon be made available through the A, B, Cs of Omega-3s eXtension website. Formative research with 352 individuals to assess their questions regarding n-3s showed consumer interest in the health benefits, food sources (and recipes), and supplements. These (and the other more specific) findings served as the foundation for the development of responses to 246 Frequently Asked Questions (FAQs) that were developed by all NC1039 members, under the leadership of the members from the New Jersey, Colorado and Nebraska Stations. The evaluation of the FAQs and additional content soon to be made available at www.extension.org, is the focus of the forthcoming proposal.

The consumption of fish and n-3 fatty acids in specific populations of Americans is not well documented. Thus, the Colorado Station studied the comparison of fish and DHA consumption by WIC participants and pregnant women in private health clinics. From the 198 food frequency questionnaires that were completed and analyzed, we learned that the WIC participants consumed 238.9+27.9 mg of DHA/day, which meets the lower end of the 200-300 mg/day recommendations for DHA in pregnant women, but is significantly less than the 300 mg/day target. When only food sources of DHA were considered an average of 133.1+21.7 mg DHA/day were consumed, which did not meet current recommendations. The total amount of DHA consumed by subjects in private health clinics was 311.0+30.6 mg/day of total DHA, which was significantly higher than total DHA intake of WIC participants. These data suggest that WIC participants consumer inadequate DHA therefore identify this group as a target population to increase DHA consumption.

Current western diets have been reported as high as 30 to 1 n-6 to n-3 ratios leading to diminished human health and increased health care costs (9). The Wisconsin Station has tested the use of a new n-3 animal feed protected supplements to use in livestock rations to improve n-3 content in food products. Additionally, supplementing livestock diets with protected n-3 fatty acids reduces veterinary related expenses, which, if extrapolated, would save Wisconsin farmers approximately $70 per head livestock per year. Other benefits to correcting n-6 to n-3 ratios in the animals feed rations are food products that are naturally 18% to 30% lower in saturated fatty acids. Thus, these innovations have great potential to impact both human and animal health through enhanced consumption of animal-derived products with higher content of n-3 fatty acids.

Objectives

  1. To determine the effects of omega-3 fatty acids on health-related outcomes in both human and rodent models.
  2. To define the mechanisms mediating the health promoting effects of omega-3 fatty acids.
  3. To evaluate the effect of an eXtension site on meeting the public's informational needs regarding omega-3 fatty acids.

Methods

Objectives for the new proposal: The problem to be addressed at that insufficient studies have been conducted into the role of n-3 fatty acids in promoting overall health and disease prevention to warrant specific recommendation and relay this information to the public. The overall goal of this application is to further characterize the functional effects of n-3 fatty acids on health-related outcomes, the mechanisms through which they exert their effects, and to provide scientific-based information via an eXtension website to the public domain. The following objectives were proposed to meet the project goal: Objective 1: To determine the effects of n-3 fatty acids on health-related outcomes in both human and rodent models. Objective 2: To define the mechanisms mediating the health promoting effects of n-3 fatty acids. Objective 3: To evaluate the effect of an eXtension site on meeting the public's informational needs regarding n-3 fatty acids. Procedures to address objective 1: Numerous studies will be carried at the Colorado Station out to test the effect of DHA intake during pregnancy and throughout exclusive breastfeeding on infant BMI in the first year of life. The overall goals of this study are to examine the effects of variable levels of maternal DHA intake during pregnancy and duration of exclusive breastfeeding on infant birth weight, growth in the first year of life, BMI and body composition and to explain infant outcomes in response to variable levels of maternal DHA by characterizing components of the mothers genome which may increase requirements for preformed DHA during pregnancy and breastfeeding. We will focus on the following specific objectives: 1) determine level of DHA intake throughout the third trimester of pregnancy and breastfeeding which promotes optimal neurocognitive development (objective of the USDA/AFRI funded study to which this study will link); 2) determine the effect of DHA and breastfeeding duration on IQ and BMI as a function of maternal genotype for 3 genes associated with the ability to convert dietary ALA to DHA and express DHA in breast milk; and 3) determine the effect of DHA and breastfeeding duration on infant adiposity using anthropometric measurements (head circumference, BMI, skinfold measurements) and air-displacement techniques (i.e. Pea Pod technology  a Colorado Agricultural Extension Station funded study to which this project will link). Additional studies at the Colorado Station aim to elucidate the influence of systemic delta 6-desaturase (D6D) activity on cardiometabolic disease risk (glucose tolerance, hyperlipidemia, inflammation, etc.) in combination with a standard low-fat diet or a high-fat/sucrose Western diet. The influence of D6D on the effect dietary essential fatty acid supplementation (LA vs. ALA) within these contexts is also an interest. To address these aims, we 1) utilize animal models of cardiometabolic disease fed experimental diets with or without treatment with an orally active D6D inhibitor, and 2) recently developed a transgenic mouse with global overexpression of D6D (fads2). Phenotyping studies of the fads2 mouse are just beginning, but once initial characterization and a stable colony are achieved, we will be eager to make this model available to members of the project for collaborative studies. We are also interested in determining the extent to which effects of exogenous DHA (obtained through dietary supplementation) differ from those elicited by endogenously produced DHA (synthesized from ALA) on the pattern of incorporation into tissue phospholipids (liver, heart, serum) and cardiometabolic risk factors. In particular, we are interested in the effect of each on endogenous liver phospholipid metabolism and how this may influence hepatic release of lipids (VLDL) into the blood. While conversion of ALA to DHA is believed to low in humans, there is accumulating evidence that common fads2 single nucleotide polymorphisms (SNPs) have significant effects on plasma D6D product/precursor ratios and cardiometabolic disease risk, indicating that variations in D6D activity may influence levels of n-3 PUFA (and other PUFAs) more than earlier studies have suggested. While an effect has been exhibited on primed (induced to ovulate) immature rats, it is unknown if rats that are allowed to ovulate on their own would have more viable offspring or if a diet enriched with n-3 fatty acids would create an ovary memory. The goal of studies proposed by the Wyoming Station would be to determine if what the rat dam consumes whiles the pups are in utero or when the pups are immature will alter ovary behavior once they mature and are allowed to naturally reproduce. Polycystic ovary syndrome is a disorder of unknown etiology or progression. Long-term studies based on preliminary data are essential on metabolism in general and behavior and metabolism of adipose tissue in response to n-3 fatty acids. In defining the mechanisms mediating the health promoting effects of n-3 fatty acids the Wisconsin Station we will further quantify the incorporation of n-3 fatty acids into livestock tissues (muscle, milk, etc.) from animals fed diets enriched in protected n-3 fatty acids and develop new food lines (cheeses, baked goods, etc.). We expect not only improved animal health, but also a final food product highly enriched in n-3 fatty acids. Additionally, foods generated for human testing will be shared with Nebraska, Wyoming, Colorado and North Dakota Stations for lipid and sensory analysis. North Dakota and Tennessee Stations will determine the effects of n-3 fatty acids on markers of adipose tissue inflammation, insulin resistance and diabetes. Specifically, they will determine if n-3 fatty acids feeding reduces adipose tissue inflammation and diabetes outcomes by decreasing adipose tissue mass, effects mediated by decreased adipocytes number, due to inhibition of adipose stem cell proliferation and differentiation into adipocytes, and decreased adipocyte hypertrophy via decreased lipogenesis. These stations have previously demonstrated that n-3 fatty acids including SDA, EPA and DHA exert anti-inflammatory activity in vitro and in vivo (10, 11). For in vivo studies, C57BL/6J mice will be fed diets containing low-fat diet, high saturated fat or high saturated fat plus different doses of EPA for 11 weeks. Tissues and serum will be collected and changes in metablolites, hormones and inflammatory markers will be assessed both at the tissue and systemic levels. In collaboration with other stations conducting animal studies, we will be studying adipose tissues collected from other n-3 fed animals. We are also interested in assessing circulating markers of inflammation in collaboration with stations conducting both animal and humans n-3 PUFA interventions. Procedures to address objective 2: The Wyoming Station will examine the effect of n-3 fatty acids on circulating hormones and adipokines from adipose tissue upon isolation and in cell culture and the specific role these metabolites and their regulatory proteins play in the PCOS and its associated reproductive infertility. As tissue can and will be shared between all participating members of multistate group these studies will allow for combined studies on reproductive issues related to PCOS, type 2 diabetes, possibilities related to the role of hormone-driven development endometrial and possibly breast cancer as well adipose tissue metabolism and obesity. Mechanistic studies focus on the effect of DHA enrichment of mitochondrial membrane phospholipids on mitochondrial function in rodent models of human disease (aging, diabetes, heart failure, ischemia and Barth Syndrome). Basic studies on the mechanisms of phospholipid remodeling, particularly DHA incorporation, in disease states are also an interest. We utilize HPLC-GC to examine the composition of individual phospholipid classes from tissue and mitochondrial samples, and cardiolipin molecular species. Additional studies are planned to evaluate the effect of supplemental and endogenous metabolism of n-3 fatty acids (ALA and DHA) on hepatic phospholipid composition, and how this may influence assembly and release of very low-density lipoprotein and subsequent risk of atherosclerosis. The studies planned at the Nebraska Station will evaluate the role of different fatty acids on the gut microbiome and characterize a potential biomarker of n-3 fatty acids. Wild-type C57BL/6J mice will be fed diets with defined fatty acid profiles enriched in saturated fat, plant-derived polyunsaturated fat, marine-derived n-3 fatty acids or a mixture of saturated fat and n-3 fatty acids from weaning to 12 weeks of age. In the present work, we will perform a metagenomic profile of gut bacteria to determine the impact of dietary fat on the gut bacterial population. Data will also be collected for the analysis of liver function, serum FA profiles, and markers of inflammation and bacterial translocation. These data are expected to support the conclusions that (i) different classes of dietary fatty acids modulate both host and gut bacterial metabolism and (ii) n-3 fatty acids specifically promote hepatic and gastrointestinal health. In previous studies we determined a lipocalin family member, LCN13, had high expression when mice were fed diets enriched in n-3 fatty acid (5). In humans, a lipocalin called odorant binding protein IIb (OBPIIb) shares 47% amino acid identity with murine LCN13. Using the human hepatocyte model cell line, HepG2, we found OBPIIb expression was induced by DHA and a n-3 fatty acid mixture that mimicked our in vivo studies. We now propose to develop an antibody based ELISA assay system to address the hypothesis OBPIIb is a biomarker for HUFA levels in humans and a biomarker for health. The human samples we will use will be plasma samples supplied by the Colorado Station from pregnant women supplemented for 8 weeks in the third trimester of pregnancy with 600 mg per day DHA or a placebo. These samples will be blinded with regard to patient identity. We expect these experiments will support the hypothesis, OBPIIB is the functional equivalent of murine LCN13 and may be used as a biomarker of n-3 fatty acid levels and associated health benefits. The North Carolina Station we are using both in vitro and ex vivo approaches to further elucidate the mechanisms by which a disruption in lipid rafts with n-3 PUFAs modifies the clustering of specific proteins involved in B cell activation. Specifically, we are studying how n-3 fatty acids manipulate the molecular organization of Toll-like receptors that are involved in responding to activation stimuli. Understanding this mechanism has profound implications for the development of strategies that can use fish oil for targeting innate immune responses in a clinical setting. N-3 fatty acids are known to impact lipoprotein metabolism through a variety of mechanisms, and are therefore used therapeutically to reduce blood triglyceride concentrations (12). How therapeutic doses of n-3 fatty acids affect cholesterol ester transfer protein-mediated lipid transfer is not clear. The Wisconsin Station propose to utilize stable isotope techniques to determine how pharmacological doses (>3 g/d) of EPA + DHA impact cholesterol ester transferase mediated lipid transfer and thus high-density lipoprotein particle composition. Hypertriglyceridemic subjects will undergo the stable isotope protocol before and after a 4-week intervention period, during which they will receive different doses fish oil supplements. Blood samples will be collected in the fasted and fed states, and lipoproteins will be isolated to determine the transfer of triglyceride from triglyceride rich lipoproteins to high density lipoproteins. The percent of triglyceride fatty acids from dietary and de novo sources will be determined using GC/MS. By determining the contribution of these fatty to high density lipoprotein triglycerides pre and post intervention, this study will help elucidate the mechanism of how n-3 fatty acids improve cardiovascular health. North Dakota and Tennessee Stations will conduct studies to investigate the mechanism by which n-3 fatty acids block adipose stem cell differentiation into adipocytes. We hypothesize that n-3 fatty acids reduces adipose stem cell differentiation into adipocytes by inhibiting gene expression, protein content and enzyme activities of target proteins. Additionally, we will use 3T3-L1 adipocytes and human adipocytes to examine signaling pathways and mechanisms mediating the anti-inflammatory effects of n-3 fatty acids. Finally, we will conduct extensive expression analysis including microarray and proteomic analyses to determine the effects of ALA, SDA, EPA, DHA, and AA on regulation of signaling pathway activation underlying cytokine and chemokine gene expression alterations in obese and diabetic mice. Procedures to address objective 3: The research that will be done via this proposal at the New Jersey, Delaware and Tennessee Stations is aimed at evaluating the A, B, Cs of Omega-3s eXtension website, and its impacts. The work will be conducted in three complimentary phases: Phase 1 (2013-14): An assessment of the website will be conducted with Extension personnel and their clientele. Participants will include Extension Family and Community Science/Nutrition Educators in Tennessee and New Jersey, as well as clientele recruited through Extension personnel. Study participants will be asked to explore the A, B, Cs of Omega-3s website and to provide feedback, i.e., quantitative (ratings) and qualitative (comments), regarding their likes and dislikes of the website during their investigation. Research computers will be used so that time spent on each of the various sections of the website will be logged using spyware. Following their exploration, researchers will review their responses and probe when necessary for clarification and additional information. A semi-structured interview will be conducted to query facets of the website that were not previously addressed; demographic information will also be collected. Station researchers hypothesize that the website format will be preferred by those with higher educational levels, but the website content will be universally favored. Researchers also predict that content will require some reorganization and modification based on user feedback. Phase 2 (2015-16): After the necessary adjustments to the website have been made, based on phase one findings, focus groups will be held with Extension Family and Community Science/Nutrition Educators to assess the best means for marketing the website to their clientele, as well as to other Extension personnel. A marketing plan for the website will consequently be devised and put into place. Phase 3 (2017): This portion of the study employs an experimental design. Members of the general public and pregnant women from WIC office waiting rooms (divided equally among control and experimental group) will be recruited. The Colorado Station will assist with the WIC subject analyses. Inclusion criteria will be those who are: " Between the ages of 18-35 " Use the internet to acquire health information " Able to speak, read and understand English Those who agree to complete the following surveys and to be contacted two months later for a follow-up survey will be recruited for study participation. Surveys and food frequency questionnaires, designed to assess subjects stage of change with regard to the consumption of n-3 fatty acid rich foods and supplements. Participants will also be asked to complete a brief demographic survey which will include questions about the subjects use of websites, Facebook, blogs and Twitter and their health history. Upon completion of the pre-test survey, participants in the experimental group will be given a business card printed with the information about how to access the eXtension website and its corresponding Facebook, blog and Twitter communications. They will be invited to try these various venues and give us feedback on which they liked best and why. A follow up semi-structured interview will be administered after two months of the initial meeting. The control group will only be asked questions regarding stage of change and to complete a food frequency questionnaire. Experimental subjects will be asked if they went to the website. If not, they will be asked why and the same surveys that were administered to the control subjects will be done. Those in the experimental group who used the website will be asked to provide feedback on the various media on the website, whether they used it or not, and their likes and dislikes, in addition to the questionnaires completed by the others. Studies will be undertaken at the North Carolina Station using food frequency questionnaires and dietary intervention to determine the levels of n-3 fatty acids present in diets within the traditional diets of individuals living in the Southeast. Based on questionnaire information, diets will be examined to identify at risk populations that may benefit from nutrition education on the healthy properties of n-3 fatty acid containing foods. These results will identify specific populations that could most benefit from the eXtension website.

Measurement of Progress and Results

Outputs

  • Data on n-3 fatty acid intakes across different populations
  • Expansion of meat and dairy products with high n-3 fatty acid levels
  • Data providing additional support for dose and type specific effects of n-3 fatty acids
  • Novel biomarkers of n-3 fatty acid intake
  • Novel mechanisms through which n-3 fatty acids influence inflammation
  • Output 6: Data on the biophysical effects of n-3 fatty acids on lipid rafts and cell signaling Output 7: Data further defining the n-3 fatty acid requirements during pregnancy Output 8: Dissemination of data at scientific conferences and publications Output 9: Webpage for dissemination of n-3 fatty acid related information to the general public

Outcomes or Projected Impacts

  • Increased awareness into the health benefits of n-3 fatty acids
  • Improved health of the US population
  • Findings that support more defined recommendations for n-3 fatty acid intake
  • Identification of populations that can be targeted to increase n-3 fatty acid intake
  • Increased n-3 fatty acid intake
  • Outcome/Impact 6: Increased terrestrial-based food products with higher n-3 fatty acid content Outcome/Impact 7: Improved assessment of n-3 fatty acid intake

Milestones

(2013): Present research findings at scientific meetings such as the American Society of Nutrition, the Obesity Society, the Society for Nutrition Education and Behavior and the International Society for the Study of Fatty Acids and Lipids

(2014): Present research findings at scientific meetings (as above)

(2015): Present research findings at scientific meetings (as above)

(2016): Present research findings at scientific meetings (as above)

(2017): Present research findings at scientific meetings (as above)

Projected Participation

View Appendix E: Participation

Outreach Plan

The ABCs of Omega-3s website as described in objective #3 is devoted to outreach.

Organization/Governance

This multistate committee comprised of scientists from participating stations, the Regional Administrative Advisor and the USDA CSREES Representative will be organized and governed as specified in the North Central Regional Association (NCRA) guidelines.

At the annual meetings, a Chair and Secretary are elected for the upcoming year. In addition, members of the Technical Committee will elect members to a committee responsible for organizing national/international presentations (see Milestones).

Literature Cited

1. IOM. 2005. Dietary reference intakes: energy, carbohydrates, fiber, fat, fatty acids, cholesterol, protein, and amino acids. Washington D.C. National Academy of Sciences Press.
2. Gebauer, S. K., T. L. Psota, W. S. Harris, and P. M. Kris-Etherton. 2006. n-3 fatty acid dietary recommendations and food sources to achieve essentiality and cardiovascular benefits. Am J Clin Nutr 83: 1526S-1535S.
3. Schwinkendorf, D. R., N. G. Tsatsos, B. A. Gosnell, and D. G. Mashek. 2011. Effects of central administration of distinct fatty acids on hypothalamic neuropeptide expression and energy metabolism. Int J Obes (Lond) 35: 336-344.
4. Sealls, W., M. Gonzalez, M. J. Brosnan, P. N. Black, and C. C. DiRusso. 2008. Dietary polyunsaturated fatty acids (C18:2 omega6 and C18:3 omega3) do not suppress hepatic lipogenesis. Biochimica et biophysica acta 1781: 406-414.
5. Gonzalez, M., W. Sealls, E. D. Jesch, M. J. Brosnan, I. Ladunga, X. Ding, P. N. Black, and C. C. DiRusso. 2011. Defining a relationship between dietary fatty acids and the cytochrome P450 system in a mouse model of fatty liver disease. Physiological genomics 43: 121-135.
6. Rockett, B. D., M. Salameh, K. Carraway, K. Morrison, and S. R. Shaikh. 2010. n-3 PUFA improves fatty acid composition, prevents palmitate-induced apoptosis, and differentially modifies B cell cytokine secretion in vitro and ex vivo. J Lipid Res 51: 1284-1297.
7. Rockett, B. D., A. Franklin, M. Harris, H. Teague, A. Rockett, and S. R. Shaikh. 2011. Membrane raft organization is more sensitive to disruption by (n-3) PUFA than nonraft organization in EL4 and B cells. J Nutr 141: 1041-1048.
8. Weldon, K. A., and J. Whelan. 2011. Allometric scaling of dietary linoleic acid on changes in tissue arachidonic acid using human equivalent diets in mice. Nutrition & metabolism 8: 43.
9. Christophersen, O. A., and A. Haug. 2011. Animal products, diseases and drugs: a plea for better integration between agricultural sciences, human nutrition and human pharmacology. Lipids in health and disease 10: 16.
10. Hsueh, H. W., Z. Zhou, J. Whelan, K. G. Allen, N. Moustaid-Moussa, H. Kim, and K. J. Claycombe. 2011. Stearidonic and eicosapentaenoic acids inhibit interleukin-6 expression in ob/ob mouse adipose stem cells via Toll-like receptor-2-mediated pathways. J Nutr 141: 1260-1266.
11. Kalupahana, N. S., K. Claycombe, S. J. Newman, T. Stewart, N. Siriwardhana, N. Matthan, A. H. Lichtenstein, and N. Moustaid-Moussa. 2010. Eicosapentaenoic acid prevents and reverses insulin resistance in high-fat diet-induced obese mice via modulation of adipose tissue inflammation. J Nutr 140: 1915-1922.
12. Adkins, Y., and D. S. Kelley. 2010. Mechanisms underlying the cardioprotective effects of omega-3 polyunsaturated fatty acids. J Nutr Biochem 21: 781-792.

Attachments

Land Grant Participating States/Institutions

CO, CT, GA, IL, MI, MO, NE, NJ, OH, SD, TN, TX, UT, WV

Non Land Grant Participating States/Institutions

East Carolina University, Laboratory / Livestock Project Integrator, NUTRITION, Tennessee State University, Texas Tech University, Texas Woman's University, University of Delaware, University of Wisconsin - Stout, USDA-ARS, USDA-ARS/Wisconsin
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