Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
Nutrition Science
Non Technical Summary
Chronic diseases and conditions are the most common and costly health problems worldwide. Chronic diseases affect approximately 50% of adults in the U.S. and account for 60% of deaths worldwide [26, 29]. Many of these diseases are preventable by healthy lifestyle choices including good nutrition [25]. For instance, increased consumption of omega-3s improves rheumatoid arthritis [22], vision [5], neurodegenerative diseases [28], cardiovascular function [3], recovery from traumatic brain injury [16], and fetal growth and development[17]. Omega-3 fatty acids confer health benefits by serving as critical components for proper membrane fluidity and by acting as anti-inflammatory mediators [19]. The recognition of omega-3s health benefits are exemplified by the recent FDA allowance of the food claim: "consuming omega-3s may reduce the risk of heart disease"[24]. The need to increase human omega-3 fatty acid consumption through regular intake of fish and seafood is recognized and recommended by many national and international organizations and agencies, including the U.S. Dietary Guidelines, Institute of Medicine, Food and Agriculture Organization of the United Nations, and World Health Organization. Markets across the globe recognize the commercial demand for increasing omega-3 fatty acids in food products. The global omega-3 market was estimated to be $1.82 billion in 2014 and is projected to grow to $10+ billion by 2022 due to increased demand [1]. Simultaneous to this increased market demand for omega-3s, is the depletion of wild fish from overfishing practices, thus the aquaculture industry has grown and has adopted more ecofriendly practices by switching from fish feed obtained from DHA-rich fish/krill oil to fish feed derived from soy, a more sustainable, but omega-3-deplete feed source. The resulting omega-3-deplete fish food products stands to severely limit human dietary consumption of omega-3, and in turn increase the risk of chronic disease. Algae are naturally omega-3 rich and the industry is experiencing rapid growth due to: 1) the ability to extract alternative and renewable biofuels from algae and 2) the production of algae-derived omega-3s human supplements and food additives. Thus, extending the use of algae-derived omega-3s to animal and fish feed represents an additional means of human dietary supplementation. In summary, the combination of omega-3-conferred health benefits against common chronic diseases, international recommendations to increase omega-3s consumption, increased market demand for omega-3s, the growing algae industry, sustainability considerations surrounding wild fish conservation, and the critical role of omega-3s to enhance health suggest that exploration of the benefits from omega-3 enhanced foods is of timely and critical importance. However, significant gaps in research remain regarding the efficacy/bioavailability of omega-3s-rich food products and the specific mechanisms though which omega-3s are metabolized and act to protect against chronic neurological disease. Here, we will address these gaps.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Our overall research goals are to discover the regulatory mechanism governing the incorporation, metabolism, and protective effects of omega-3 fatty acids in the central nervous system.Assess methods to increase omega-3 fatty acids in the human food supplyGoal: Fish are the primary dietary source of DHA because of DHA-rich algae in their food-chain. However, with the depletion of fish species from overfishing, the aquaculture industry has grown and adopted more ecofriendly practices involving the use of sustainable soy as the source of fish feed. Unfortunately soy is DHA-deplete and the resulting DHA-deplete farmed fish stands to severely limit human dietary DHA consumption, and in turn, increase the risk of chronic disease. Thus, there is a need to increase DHA in the diet using the most bioavailable means. We propose to work with our collaborators to obtain foods (fish and beef) that have been DHA-enhanced and feed them to mice to determine their ability to enhance DHA content, compared to DHA supplements, in the brain of consumers.Determine the mechanisms regulating omega-3 fatty acid incorporation in the central nervous systemGoal: The regulatory mechanisms governing omega-3 metabolism in the central nervous system remain elusive. We will study a central nervous system-enriched, omega-3-preferring enzyme, acyl-CoA synthetase 6 (Acsl6) and its role in the incorporation and metabolism of dietary omega-3s within the central nervous system. To study this, we have generated a novel, Acsl6 knockout mouse model. We have preliminary data to show that DHA is specifically reduced in the brain of Acsl6 knockout mice. We will further investigate this mouse model to determine how Acsl6 regulates incorporation and downstream metabolism of omega-3s in the central nervous system.Determine the role of omega-3 fatty acid metabolism in neurological healthGoal: We will use our novel Acsl6-deficient mouse model and specialized diets to establish the role of and mechanisms through which omega-3 metabolism protects against disease in the central nervous system.
Project Methods
Determine ability of omega-3-enhanced foods to improve tissue omega-3 lipid profiles.For this objective, we will obtain omega-3-enriched and control (non-enriched) foods from our collaborators in the College of Agriculture at Purdue and feed these foods to mice to determine the ability of omega-3-enhanced foods to improve lipid content across tissues.Procurement of omega-3-enriched foods from our collaborators:Fish: Salmon will be raised at the Purdue University Aquaculture Facility by Dr. Paul Brown. Fish will be fed experimental diets: 1) fishmeal-based, 2) soy-based, or 3) soy + algae-omega-3 for 12 weeks [2].Cattle: Steers will be raised in the Beef Unit of the Animal Sciences Research and Education Center (ASREC) of Purdue University by Dr. John Schoonmaker. Cattle will be randomly assigned to diet groups: 1) control with no algae meal or 2) supplemental omega-3 rich algae meal top-dressed. Animals will be slaughtered at Purdue and processed into ground beef product [27].Effect of foods on tissue lipid profiles:Mouse Studies: Mice will be fed chow diets and split into 4 groups: 1) control; 2) algae-derived oil supplement; 3) control fish and beef from soy or grain fed animals; or 4) DHA-enhanced fish and beef from algae-omega-3 fed animals. The mice will have free access to fish and beed 3 nights/week or gavaged with omega-3 oil 3 times/week. We will order male C57Bl/6J mice from Jackson labs for these studies and they will be randomly assigned to the diets. After 4 weeks of dietary treatment, we will harvest tissues and run omega-3 lipidomics analysis on brain, muscle, and serum.Determine the mechanisms regulating omega-3 fatty acid incorporation in the central nervous system. For this objective, we will use our novel Acsl6 knockout mouse which we have discovered is specifically deficient in brain DHA content with concomitant increase in omega-6 fatty acid content. We will use this model to determine the role that Acsl6 plays in regulating the uptake and incorporation of lipids into the brain.Generation of Acsl6 deficient mice: Acsl6 floxed mice were created by Ingenious Targeting, Inc and bred to the ubiquitous and germline deleted Cre-line, CMV-Cre. We have shown that ACSL6 protein is absent in the central nervous system mice and that total enzyme activity for acyl-CoA synthetases is reduced by 40% for the omega-3 fatty acids.Lipidomics and metabolomics: We are in the process of collecting lipidomics and metabolomics data from control and Acsl6 knockout brains with our collaborator Dr. Dan Nomura of UC-Berkley. In addition we will perform lipidomics analysis to screen for specific omega-3 and omega-6 downstream bioactive metabolites.Lipid flux determination: We will determine how Acsl6 regulates lipid flux by performing radiolabel tracer experiments on brain slices from control and Acsl6 knockout mice. Whole brains will be sliced using the McIlwain tissue chopper and incubated with radiolabeled substrates. The metabolic fate of these substrates will be determined using methods we have previously described [12].Membrane fluidity and potential assessment: To determine how Acsl6 deficiency alters membrane properties, we will measure membrane fluidity and membrane potential in ex vivo brain slices using the membrane fluidity kit and cellular membrane potential assay kit (abcam).Determine the role of omega-3 fatty acid metabolism in neurological health. For this objective we will use our Acsl6 knockout mice to better understand the role of omega-3 fatty acids in neurodegeneration, neuroprotection, and behavior. We will age mice up to 18 months and the following experiments will be performed:3a. Neurodegenerative assessment:There are strong associations between increasing omega-3 consumption to improve many aspects of aging and to prevent neurodegenerative disease. Here, we will use several methods to assess neurodegeneration to determine how Acsl6 and omega-3 metabolism in the central nervous system influence aging and other neurodegenerative outcome.Pre-pulse inhibition: Acoustic startle response/prepulse inhibition will be measured in startle chambers in collaboration with Dr. Julia Chester. Pre-pulse inhibition will be assessed across 5 prepulse intensities at 20ms each, with variable combinations of exposure and after an acclimation period, as we have previously described [12].Motor coordination: Motor coordination on a rotarod treadmill will be assessed by a blinded investigator. Mice will be rotarod trained and experimental data are obtained based on the average time spent on the rod over three tests as the rod rotation accelerates from 4 rpm to 40 rpm over 500 s.Cerebellar ataxia: Ataxia is screened by a blinded investigator for four phenotypic measures, hind limb clasping, ledge test, gait, and kyphosis. Ataxia test values are calculated by the average for three trials, in which mouse performance is scored between 0 to 3 for best to worst performance, as described [12].Molecular assessment of age-related neurodegeneration: Upon tissue collection, mouse brains will be assessed for molecular indicators of neurodegeneration by western blot, mRNA quantification, and histological indicators of reactive gliosis and neuroinflammation.3b. Neuroprotection assessment:Due to the connection between omega-3 intake and reduced neurodegeneration, we predict that omega-3 fatty acids are neuroprotective. To test this, we will perform several experiments on control and Acsl6 knockout mice to determine the role of Acsl6 and omega-3s on neuroinflammation.Lipopolysaccharide challenge: E. coli lipopolysaccharide (serotype 0127:B8, sigma) or saline vehicle will be injected i.p. at a dose of 0.33 mg/kg body weight. Mice will be immediately placed into the Laboratos chambers, in collaboration with Dr. Diane Little, for continual in-cage behavior and activity monitoring for 24 hours. After which tissues will be harvested for assessment of neruoinflammatory response by nuclear localization of NFkB; induction of mRNA markers of inflammation (Il-1beta, TNFalpha, Il-10, Il-6, etc.); oxidative stress by TBARS assay; and indicators of lipid-derived signaling molecules by targeted lipidomics, expression of cyclooxygenase enzymes, and activity of phospholipase A.Retinopathy: We will harvest retinas from control and Acsl6 knockout mice to assess molecular markers of retinopathy. This work will be performed in collaboration with Dr. Teri Beleckey-Adams, IU. We will also assess vision using a cliff test and hanging bar test.3c. Neurobehavioral assessment:Several reports have linked Acsl6 to schizophrenia and addiction behaviors. We will assess behavior and addiction in control and Acsl6 knockout mice with the following assays: Fear conditioning: Fear-potentiated startle will be assessed in collaboration with Dr. Julia Chester. Animals will be placed in Animal Acoustic Startle System chambers and exposed to combinations of foot shock, light, and acoustic stimuli in a manner that will assess fear-propitiated startle, as previously described [14].Addiction behavior/Alcohol preference: Alcohol preference will be determined in collaboration with Dr. Julia Chester. Mice will be given free choice between tap water and a 10% alcohol solution in tap water under 24-h access conditions with rotating cylinder position. Fluids will be weighed across the course of the experiment to determine preference.Dietary omega-3 influence on Acsl6-mediated neurological outcomes: Once we established the ability DHA-enhanced foods to increase omega-3 lipid content in mice, we will feed our control Acsl6 deficient mice this diet and repeat experiments where stark genotypic differences were observed to determine if Ascl6 is required for dietary omega-3 fatty acid conferred protection.