Location: Immunity and Disease Prevention Research2016 Annual Report
Objective 1: Determine the differential metabolism and uptake of n-3 fatty acids from dietary phospholipid and triglyceride forms in different tissues. -Sub-objective 1A: Determine the differential uptake and accretion of PL and TG forms of DHA in different mouse tissues. - Sub-objective 1B: Compare the metabolism of PL and TG forms of DHA in different mouse tissues. - Sub-objective 1C: Determine whether the differential accretion of DHA in brain from the PL and TG forms will result in differences in mouse behavior. Objective 2: Compare the anti-inflammatory and immune-modulating ability of n-3 fatty acids from dietary phospholipid and triglyceride forms in different tissues. -Sub-objective 2A: Compare the anti-inflammatory and immune-modulating ability of PL and TG forms of DHA by using experimental allergic encephalopathy (EAE)as a mouse model of brain inflammation. - Sub-objective 2B: Compare the anti-inflammatory and immune-modulating ability of PL and TG forms of DHA in the prevention of diet-induced NAFLD in mice.
Proposed experiments will compare accretion, metabolism and health effects n-3 fatty acids from the phospholipid (PL) and the triglyceride (TG) forms in different mouse tissues. For objective 1, we will determine the dose dependent accretion and metabolism of the PL-DHA and TG-DHA in the mouse tissues and their effects on the behavioral responses. We hypothesize that tissue accretion of DHA will increase with its increased intake, and at a given dose it will be greater in the PL group than in the TG group for tissues which express mfsd2a transporter (brain and liver) and will be similar in tissues (heart, muscle, and adipose tissue) that do not express this transporter. We also hypothesize that concentration of DHA in different lipid classes and PL subclasses, and that of DHA metabolites will be a function of tissue DHA concentration, and that DHA will improve learning and memory, and decrease fear and anxiety induced stress; effects of PL-DHA will be greater than that of TG-DHA on the behavioral responses tested. For objective 2, we will compare anti-inflammatory and immune-modulating effects of single equivalent dose (based on the results of objective 1) of the PL- and TG-DHA on brain and liver. For studies with brain we will use the mouse model of experimental allergic encephalomyelitis (EAE), and for liver we will use the high fat and high sucrose diets fed mouse model of nonalcoholic fatty liver disease (NAFLD). We hypothesize that both forms of DHA will delay the onset and severity of EAE and NAFLD when compared with the control groups, and PL-DHA will be more effective than TG-DHA. We anticipate our results will support the proposed hypotheses, however, if DHA does not have any effect on the response variables tested, we may repeat some experiments by increasing the duration of feeding. Whether or not the two forms of DHA differ in their effects on the responses tested will be equally useful information. A variety of behavioral, biochemical, molecular, immunohistochemistry, flowcytometery, and other analytical methods will be used. The mouse models and the methods proposed here have been previously used in our or our collaborator’s laboratories.
This report period covers the last half of project year one and the first half of project year two. A key component of this project was securing highly purified phospholipid docosahexaenoic acid (PL-DHA) and triglyceride docosahexaenoic acid (TG-DHA) in quantities sufficient for conducting feeding studies in mice. Manufacturing went more slowly than expected and these materials were received in January, 2016. The next step was to determine the best method for incorporating these lipids into the powdered diet, which was then formed into pellets and to determine the need for pair feeding. After testing different methods, due to unexpected physical characteristics of the PL-DHA, this step was completed in March 2016. Finally, we conducted a small animal experiment to determine if the two diets were accepted similarly by mice. If they were not, differential intake could affect the outcome of the study. Fortunately the mice did not show any difference in food intake with the two diets. Different amounts of food consumed by different dietary groups would have required pair feeding the mice in the future studies. The results of this preliminary study avoided the need for pair feeding. Thus we completed the “plan and prepare” component of Objective 1 during this reporting period. The experimental studies proposed under Sub-Objectives 1A, 1B, and 1C were started in April 2016 and data collection for food intake, body weights, behavioral tests and tissue collection were completed in June 2016. Laboratory analysis of tissue samples from this study is in progress. It will involve analysis for fatty acids, other lipids and their metabolites, protein and mRNA for fatty acid binding and transport proteins from livers, brain, heart, adipose tissue and muscle from seven dietary groups. Statistical analysis of these data will follow completion of their laboratory analysis. Beginning subsequent mouse studies (described in Sub-Objective 2A) depends on the results from Objective 1. The studies under Objective 1 compared different dietary levels of PL-DHA and TG-DHA and the dietary studies in Objective 2 will use one of these doses (the one showing the most obvious differences in tissue levels of DHA when comparing the PL and TG forms used in the diets). Thus completion of laboratory work and related data analysis is needed in order to plan the diets for Objective 2.