Modulation of Adipocyte Function by Dietary Omega-3 Fatty Acids Carla Taylor, PhD Departments of Human Nutritional Sciences and Physiology, University of Manitoba & Canadian Centre for Agri-food Research in Health & Medicine, St. Boniface Research Centre

The Omega-3 Fatty Acid Family α-Linolenic acid (ALA) 18:3 n-3 Eicosapentaenoic acid (EPA) 20:5 n-3 Nomenclature: Omega-3’s are fatty acids 18 carbons or longer with two or more double bonds, where the first double bond occurs at the 3rd carbon from the omega end (or the carbon end) of the hydrocarbon chain. These are the three best-studied members of the omega-3 family: ALA, EPA and DHA. These are essential fatty acids, meaning that they must be obtained in the diet because humans lack the enzymes required to synthesize them. Dietary sources of ALA are canola and flaxseed, as well as other plant-based products such as almonds and walnuts, hempseed and chia seeds. EPA and DHA are mainly obtained from fish, such as cod, mackerel, tuna or salmon. The fish obtain the omega-3’s from the algae that they consume. Much of the research on omega-3 fatty acids has focused on the latter two fatty acids, while ALA has been largely overlooked. Docosahexaenoic acid (DHA) 22:6 n-3

Polyunsaturated Fatty Acid Metabolism Omega-3 Fatty Acids Omega-6 Fatty Acids Alpha-linolenic acid ALA 18:3 n-3 Linoleic acid LA 18:2 n-6 Δ6 desaturase Stearidonic acid 18:4 n-3 Gamma-linolenic acid GLA 18:3 n-6 elongase δ-6-des Eicosatetraenoic acid 20:4 n-3 Dihomo γ-linolenic acid DGLA 20:3 n-6 Δ5 desaturase Eicosapentaenoic acid EPA 20:5 n-3 Arachidonic acid AA 20:4 n-6 elongase Docosapentaenoic acid DPA 22:5 n-3 Adrenic acid 22:4 n-6 ALA is a precursor to EPA and DHA, so when you consume a food that contains ALA, there could be biological activity from the ALA itself, or through conversion to EPA and DHA. Speaking of biological activity – The omega-3 elongation and desaturation pathway runs in parallel to the omega-6 fatty acid pathway, and they share and compete for most of the same enzymes. In a typical Western diet, omega-6 intake far exceeds omega-3 intake, and so the omega-6 fatty acids pathway is more dominant and the by-products of this pathway (for example, AA) are much more prevalent. AA is a precursor to a series of prostaglandins, thromboxanes and leukotrienes that are highly inflammatory. While EPA on the omega-3 side also leads to the production of prostaglandins and others, these tend to be much less inflammatory or inflammation-modulating. This is one of the proposed mechanisms for the anti-inflammatory activity of omega-3 fatty acids. elongase ω3 Tetracosapentaenoic acid 24:5 n-3 ω6 Tetracosatetrenoic acid 24:4 n-6 Δ6 desaturase ω3 Tetracosahexenoic acid 24:6 n-3 ω6 Tetracosapentaenoic acid 24:5 n-6 peroxisomal beta-oxidation Docosahexaenoic acid DHA 22:6 n-3 ω6 Docosapentaenoic acid 22:5 n-6 Adapted from Sprecher (2000)

Substrates for Signalling Molecules Receptor-mediated Signalling Physiological Roles of Omega-3 Fatty Acids Membrane Components Substrates for Signalling Molecules Receptor-mediated Signalling Membrane fluidity Protein localization Ion channel function Signalling events Eicosanoids Prostaglandins Leukotrienes Endocannabinoids Extracellular and Nuclear Receptors Other biological or physiological roles for omega-3 fatty acids include their incorporation into the cell membrane, where they can affect membrane fluidity, protein localization and ion channel function. This is the proposed mechanism for possible anti-arrhythmic effects in the heart. They can also act as substrates for signalling molecules, such as inflammatory mediators. Omega-3’s can also bind cell membrane and nuclear receptors to trigger signalling cascades and affect gene expression, and it’s believed that this mechanism also helps to modulate inflammation. Overall, these biological actions are thought to reduce mortality from cardiovascular disease. Various Organs

Adipose tissue an endocrine organ Dysfunctional Adipose tissue Healthy Adipose tissue Macrophage Obesity Blood vessele Adiponectin Omentin Unlike many people think, fat tissue is not just a fat storage organ. fat tissue is one of the important endocrine organs in our body, which actually secretes hormones that have many beneficial effects. these hormones are called adipokine The level of these hormones is dependent on the fat cell status. Healthy small adipocytes secrete hormones such as adiponectin and omentin, which are anti-inflammatory and their cardio protective effect is well known, The problem is when these small healthy fat cells get enlarged and became unhealthy, which is the case during obesity. So these unhealthy fat cells produce less adiponectin and instead they Secrete the hormones that are known to be pro-inflammatory and this leads to macrophage infiltration in fat tissue. This altered adipokine profile and macrophage infiltration leads to the complications of obesity. So all in all Visfatin Vaspin Resistin Leptin Chemerin

Research Question: What are the effects of dietary intervention with plant-based omega-3 fatty acids (ALA) on adipocyte size and adipose tissue function in an obese model?

Rats were 25 week old at end of dietary treatments Experimental Design 17 week old lean (ln) Zucker rats 17 week old fa/fa (fa) Zucker rats 8 weeks 8 weeks Baseline (faBASE, n=7) Control diet (lnCTL, n=7) Control diet (faCTL, n=7) Omega-3 fatty acid diet (faALA, n=7) Rats were 25 week old at end of dietary treatments

ALA Source: http://www.canola-council.org/properties.html

Fatty acid profile of the experimental diets Both diets contained 8.5 g fat/100 g diet 50:50 Flaxseed oil & soybean oil Soybean oil LA ALA n6/n3 ratio *g/100 g total fatty acids

Body weight, feed intake and obesity were unchanged Weekly body weights (g) Visceral adipose depots (g/100 g bwt) b b b a Weekly feed intake (g) Columns with different letters are Significantly different (P<0.05)

The faALA group had smaller adipocytes

The faALA group had lower levels of leptin in adipose tissue Adiponectin = Anti-inflammatory adipokine Leptin = Pro-inflammatory adipokine

The faALA group had lower levels of IL-10 & TNF-α in adipose Interleukin-10 (IL-10) = Anti-inflammatory cytokine Tumor necrosis factor-α (TNF-α) = Pro-inflammatory cytokine

The faALA group had a 5-fold reduction in adipose MCP-1 (to lean levels) MCP-1 = monocyte chemoattractant factor-1

Macrophages infiltrating adipose tissue were unchanged.

The faALA group had fewer T-cells in adipose tissue

Summary Plant-based omega-3 (ALA) in fa/fa Zucker rat model: 5-fold reduction in MCP-1 in adipose tissue 75% lower levels of TNF-α and IL-10 in adipose tissue less T-cell infiltration in adipose tissue smaller adipocyte size despite no change in adipose mass or body weight. Next Steps Comparison of plant-based omega-3 (ALA) with marine-based omega-3 (EPA vs DHA) in fa/fa Zucker rat model Oxylipins and 3T3-L1 adipocytes

Polyunsaturated Fatty Acid Metabolism “Less” inflammatory Omega-3 Fatty Acids Omega-6 Fatty Acids “More” inflammatory Alpha-linolenic acid ALA 18:3 n-3 Linoleic acid LA 18:2 n-6 Δ6 desaturase Stearidonic acid 18:4 n-3 Gamma-linolenic acid GLA 18:3 n-6 elongase Oxylipins Eicosatetraenoic acid 20:4 n-3 Dihomo γ-linolenic acid DGLA 20:3 n-6 Δ5 desaturase Eicosapentaenoic acid EPA 20:5 n-3 Arachidonic acid AA 20:4 n-6 elongase Docosapentaenoic acid DPA 22:5 n-3 Adrenic acid 22:4 n-6 ALA is a precursor to EPA and DHA, so when you consume a food that contains ALA, there could be biological activity from the ALA itself, or through conversion to EPA and DHA. Speaking of biological activity – The omega-3 elongation and desaturation pathway runs in parallel to the omega-6 fatty acid pathway, and they share and compete for most of the same enzymes. In a typical Western diet, omega-6 intake far exceeds omega-3 intake, and so the omega-6 fatty acids pathway is more dominant and the by-products of this pathway (for example, AA) are much more prevalent. AA is a precursor to a series of prostaglandins, thromboxanes and leukotrienes that are highly inflammatory. While EPA on the omega-3 side also leads to the production of prostaglandins and others, these tend to be much less inflammatory or inflammation-modulating. This is one of the proposed mechanisms for the anti-inflammatory activity of omega-3 fatty acids. Oxylipins elongase ω3 Tetracosapentaenoic acid 24:5 n-3 ω6 Tetracosatetrenoic acid 24:4 n-6 Δ6 desaturase ω3 Tetracosahexenoic acid 24:6 n-3 ω6 Tetracosapentaenoic acid 24:5 n-6 peroxisomal beta-oxidation Docosahexaenoic acid DHA 22:6 n-3 ω6 Docosapentaenoic acid 22:5 n-6 Adapted from Sprecher (2000)

Oxylipins COX PGE2 5-LOX 5-HETE Arachidonic acid 20:4 n-6 15-LOX Linoleic acid 18:2 n-6 α-Linolenic acid 18:3 n-3 9-HOTrE 9-HODE 13-HODE 15-HETE 5-HETE PGE2 5-LOX 15-LOX COX Oxylipins

INS INS, DEX, IBMX 3T3-L1 Pre-adipocyte Confluence Oxylipins (30 nM) Day 2 INS Mitotic clonal expansion INS, DEX, IBMX Day 0 Growth arrested 3T3-L1 Pre-adipocyte Confluence Oxylipins (30 nM) 2-3 Days 2 Days Oxylipins (30 nM) Oxylipins (30 nM) Day 8 Fully differentiated Day 6 Day 4 Start of differentiation Harvest For answering these objectives I am using cell culture model. in My system I am using 3T3-L1 pre-adipocytes, an established mouse cell line. By adding a stimulation cocktail, these cells will fully differentiate to adipocytes in 8 days. The CLA treatments are done at the time of stimulation and every time that the media is changed. Experiments were terminated by day 8. Oxylipins (30 nM)

affects differentiation of 3T3-L1 adipocytes. preadipocyte adipocyte PGE2 15-HETE 13-HODE 5-HETE 9-HODE 9-HOTrE Oxylipin treatment affects differentiation of 3T3-L1 adipocytes. AA  PGE2 5-HETE  AA AA  15-HETE 9-HODE  LA LA  13-HODE 9-HOTrE  ALA

inhibit lipid droplet formation & perilipin (PLIN) Lipid droplets Oxylipins from AA (5- and 15-HETE), LA (9- and 13-HODE), and ALA (9-HOTrE): inhibit lipid droplet formation & perilipin (PLIN) inhibit fatty acid synthase (FAS) affect lipid metabolism: hormone sensitive lipase (HSL) adipose triglyceride lipase (ATGL) *, significantly different from preadipocytes; #, significantly different from vehicle

Inflammatory markers: PGE2 from AA (omega-6 fatty acid) and 9-HOTrE from ALA (omega-3 fatty acid) reduced ACRP (adiponectin), an anti-inflammatory marker 5-HETE from AA (omega-6 fatty acid) reduced TNFα (tumor necrosis-alpha), a pro-inflammatory marker *, significantly different from preadipocytes #, significantly different from vehicle

Summary First comparison of bioactivity of LA and ALA oxylipins versus classical AA oxylipins Important physiological effects Oxylipins derived from omega-6 fatty acids are not necessarily pro-inflammatory and those from omega-3 fatty acids are not necessarily anti-inflammatory Focus on adipocytes & implications for obesity

Trainees Maria Baranowski Jennifer Enns Shannon Neumann Leslee Tworek Collaborators Peter Zahradka Harold Aukema Funding & Scholarships Agri-food Research Development Initiative Natural Sciences and Engineering Research Council Manitoba Health Research Council Manitoba Institute of Child Health NSERC CREATE FAST program

Thank you

Obesity fa/fa rat Lean rat + ↑adipocyte number Adipogenesis Adipose tissue mass = ↑adipocyte size (hyperplasia) + ↑adipocyte number (hypertrophy) fa/fa rat Lean rat As I mentioned obesity is an increase in adipose tissue mass, and this is results of an increase in adipocyte size (hypertrophy) These are the sections of adipose tissue from lean and obese fa/fa rats, which clearly shows the fa/fa rat has more of the larger adipocytes. Another factor that plays an important role in increasing fat mass, and the focus of my talk, is increase in fat cell number and that is what we referred to as adipogenesis One of the current research controversies in the obesity area is the role of adipogenesis in obesity. A. Noto et al. / Metabolism Clinical and Experimental 56 (2007) 1601–1611