1. 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

2. 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

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)

4. 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

5. 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

6. 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?

7. 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

8. ALA
Source:

9. 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

10. 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)

11. The faALA group had smaller adipocytes

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

13. 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

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

15. Macrophages infiltrating adipose tissue were unchanged.

16. The faALA group had fewer T-cells in adipose tissue

17. 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

18. 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)

19. 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

20. 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)

21. 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

22. 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

23. 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

24. 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

25. 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

26. Thank you

27. 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