Research Project: Metabolism and Health Effects of Phospholipid and Triglyceride Forms of Omega-3 Polyunsaturated Fatty Acids

Location: Immunity and Disease Prevention Research

2017 Annual Report

Objectives
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.

Approach
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.

Progress Report
We conducted a study in FY 2016 in which female mice were fed one of the 7 experimental diets comparing effects of the triglyceride (TG) form of docosahexaenoic acid (DHA) to the phospholipid (PL) form of DHA (control diet, 1%, 2%, 4% TG-DHA and 1%, 2%, 4% PL-DHA) for 5 weeks. Behavioral tests were conducted during the fifth week and animals were anesthetized to collect tissues at the end of that week. In FY 2017 we analyzed the behavioral data from the mouse study that was completed and a manuscript is under preparation for submission for publication. We found dose-dependent effects of DHA consumption on behavior and metabolism. DHA consumption positively influenced associative, fear-based learning, but consuming higher doses of DHA appear to induce a metabolic profile consistent with inflammation, increased spleen and liver size, and reduced body weight. Consuming comparatively smaller amounts of DHA, derived from PL but not TG, may confer the greater efficacy for DHA-dependent effects, particularly on learning behavior, and these effects may come with less negative side effects, such as inflammation. We have analyzed the fatty acid compositions of the brain and liver tissues collected from that mouse study. There were no differences in the sum of omega-6 polyunsaturated fatty acids (n6 PUFA), the sum of n3 PUFA, or the n6:n3 ratio in brain lipids, when comparing the PL- and TG-DHA treatments to one another at each DHA dose level. Nor did the brain DHA concentration differ between the control and any of the six DHA groups. However, the sum of n6 PUFA and the n6:n3 ratio for brain lipids were both significantly lower in each of the 6 DHA groups than in the control group. The sum of n3 PUFA was significantly greater in the 2 and 4% TG-DHA than in the control group. The changes in liver fatty acid profile caused by DHA compared with the control group were generally similar to those in the brain with one exception: when the effects of the two forms of DHA were compared in the liver, both the concentration of DHA alone, and the sum of n3 PUFA concentrations, were significantly greater in the TG-DHA groups than in the PL-DHA groups. A mouse study to determine the effects of the two forms of DHA on experimental autoimmune encephalomyelitis (EAE) was also proposed for FY 2017. A ten week study using 5 groups of mice (control, 0.3 and 1.0% each of the PL- and TG-DHA) is in progress and will be completed in FY 2017. All animals will be fed a control diet for 2 weeks and then divided into 5 groups (n=12) and fed the experimental diets for 8 weeks. Behavioral responses will be tested after feeding the experimental diets for 4 weeks. EAE will be induced after the completion of behavioral tests and will be monitored for 4 weeks after which the animals will be anesthetized and tissues collected.

Accomplishments
1. Dietary N-3 PUFA dampen the pro-inflammatory effects of trans fatty acids. Dietary trans fatty acids such as conjugated linoleic acid (CLA) enhance inflammation while n3 polyunsaturated fatty acids (PUFA) such as docosahexaenoic acid (DHA) reduce inflammation. However, the interaction between these two classes of fatty acids when consumed together is poorly understood, which leads to a lack of clarity when formulating dietary advice on consumption of these types of dietary fat. To address this problem, ARS scientists in Davis, California conducted a dietary study in mice and found that feeding a diet containing trans-10, cis-12 CLA significantly increased the hepatic production of pro-inflammatory oxylipins, as expected, but also found that concomitant feeding of the n3 PUFA DHA blocked this increase. This concomitant feeding of DHA with CLA also increased the production of anti-inflammatory oxylipins. Thus, the pro-inflammatory effects of the trans fatty acid CLA can be suppressed by increasing the consumption of n3 PUFA, which helps broaden the recommendation that increasing DHA consumption can dampen inflammation under different dietary conditions.

Review Publications
Adkins, Y.C., Belda, B.J., Pedersen, T.L., Mackey, B.E., Newman, J.W., Kelley, D.S. 2017. Dietary docosahexaenoic acid and trans-10, cis-12-conjugated linoleic acid alter oxylipins profiles in mouse adipose tissue. Lipids. 52(5):399-413.
Zunino, S.J., Storms, D.H., Freytag, T.L., Adkins, Y.C., Bonnel, E.L., Woodhouse, L.R., Breksa III, A.P., Manners, G.D., Mackey, B.E., Kelley, D.S. 2016. Dietary supplementation with purified citrus limonin glucoside does not alter ex vivo functions of circulating T lymphocytes or monocytes in overweight/obese human adults. Nutrition Research. 36(1):24-30.