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THE ROLE OF BOTANICAL OILS ENRICHED IN FADS2-DERIVED N-3 VS. N-6
POLYUNSATURATED FATTY ACIDS IN PREVENTION OF ATHEROSCLEROSIS
BY
SWAPNIL VIJAY SHEWALE
A Dissertation Submitted to the Graduate Faculty of
WAKE FOREST UNIVERSITY SCHOOL OF ARTS AND SCIENCES
in Partial Fulfillment of the Requirements
for the Degree of
DOCTOR OF PHILOSOPHY
Physiology and Pharmacology
AUGUST 2015
Winston Salem, North Carolina
Approved By:
John S. Parks, Ph.D., Advisor
Martha Alexander-Miller, Ph.D., Chairman
Michael C. Seeds, Ph.D.
Kylie Kavanagh, Ph.D.
Ann Tallant, Ph.D
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DEDICATION
This dissertation is dedicated to my very loving and sweet grandmothers: Manjulabai
Kashinath Shewale (paternal) from Takali, India and Janakibai Ramchandra Hire (maternal)
from Malegaon, India. My maternal and paternal grandparents were particularly keen on
providing education to their children despite all odds. My largely joint family of 20 some uncles
and aunts, parents: Vidhya and Vijay Shewale and in-laws: Jayashri and Sharad Sonawane
have encouraged all of us, including me and my loving and beautiful wife- Poonam, my brother-
Himanshu, my sister- Swati and ~30-50 some cousins to embrace similar ideologies and to
pursue our interests. My grandmothers to me are the silent sufferers and reformers of our lives.
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ACKNOWLEDGEMENTS
First and foremost, I would like to thank Dr. John S. Parks for his guidance, support,
patience and encouragement throughout the last five years of training. I can recall many-many
occasions when I may have made mistakes, asked redundant questions or spoken “too-much,
too-soon”. However, Dr. Parks has always been patient with me “to get it together” and to
progress as an advanced PhD candidate. It’s his originally well thought-out Botanical grant,
collaborations with other established PI’s, and allowing me to navigate my way through has
made this dissertation possible. I would also like to thank Dr. Rudel for his rare but extremely
insightful comments, advice and for putting the original Jornal Club, Seminar and Guest-speaker
series together. In the Lipid Sciences Department, there always was something to learn from
every presenter every week. I am extremely fortunate and grateful to (family) members of the
Parks lab and the Lipid Sciences Department. Everyone on the “Lipid” floor welcomed, helped
and taught me everything I could learn during my PhD here.
I can’t forget to thank other members from the Lipid group: Elena, Kaiser, Martha,
Matt, Xuewei, Amanda-Mark, and Xin. Elena made everything so easy like every mom does and
also saved my life once. Amanda-Mark allowed me to stay in their vacant house when I was
temporarily homeless. I would also like to specially thank Dr. Miller, for her guidance with
design, execution and interpretation of flow-cytometry related experiments, Dr. Seeds for
insights, and encouragement and Dr.’s Tallant and Kavanagh for their support, critique and
evaluation of my progress as a researcher. I would also like to thank my previous mentor Dr.
Mariana Morris for “pushing” me to accept the offer from Wake. The Department of Physiology
and Pharmacology not only accepted me but encouraged me to foster, explore and contribute in
scientific, teaching, business development and social areas as their brand ambassador. I am
eternally grateful for this experience and journey which has allowed me to look forward to my
future with optimism.
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TABLE OF CONTENTS
LIST OF FIGURES AND TABLES………………………………...……….…….…............. V
LIST OF ABBREVATIONS…………………………………………………..…….………..... VIII
ABSTRACT........................................................................................................................ XI
CHAPTER:
I. INTRODUCTION..................................................................................................1
II. BOTANICAL OILS ENRICHED IN N-6 AND N-3 FATTY ACID PRODUCTS OF
FADS2 ARE EQUALLY EFFECTIVE IN PREVENTING ATHEROSCLROSIS AND
HEPATOSTEATOSIS IN MICE .............................................................................52
III. IN VIVO ACTIVATION OF LEUKOCYTE GPR120 BY POLYUNSATURATED FATTY
ACIDS HAS MINIMAL IMPACT ON ATHEROSCLROSIS IN LDLrKO MICE......102
IV. DISCUSSION...................................................................................................166
CURRICULUM VITAE.......................................................................................................185
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LIST OF FIGURES AND TABLES
CHAPTER I:
Figure 1. Outline of lipoprotein transport and metabolism ……………………………………....9
Figure 2. Outline of biosynthesis and metabolic pathways for polyunsaturated fatty acids …24
Table 1. Classification of circulating lipoproteins………………………………………………….5
Table 2. Major enzymes important to lipoprotein metabolism ………………………………….10
Table 3. Mouse models of atheroscleroses ………………………………………………………20
CHAPTER II:
Figure 1. Body weight gain and terminal liver/body weight ratios……………………………….91
Figure 2. RBC fatty acid (FA) composition. ……………………………………………………....92
Figure 3. Percentage fatty acid composition of plasma and liver lipids………………………....93
Figure 4. Plasma lipid concentrations……………………………………………………………...94
Figure 5. Plasma lipoprotein cholesterol distribution……………………………………………...95
Figure 6. Hepatic VLDL-TG secretion rate. ……………………………………………………….96
Figure 7. Hepatic response to atherogenic diets. ………………………………………………...97
Figure 8. Aortic atherosclerosis. …………………………………………………………………...98
Figure 9. Mouse atherosclerotic plaque oxidized cholesteryl ester analysis. ……………….....99
Figure 10. LPS-stimulated eicosanoid release from peritoneal macrophages. ……………….100
Figure 11. Macrophage inflammation, foam cell formation, and chemotaxis. ………………...101
Table 1. Atherogenic Diet (AD) percentage fatty acid composition (%FA) and percentage total
energy equivalence (% EE) of individual fatty acid…………………………………………….….89
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CHAPTER III:
Figure 1. L-GPR120 does not affect plasma lipids…………………………………………...131
Figure 2. L-GPR120 does not affect plasma lipoprotein cholesterol distribution…………..132
Figure 3. Effect of L-GPR120 on hepatic M1-M2 and lipogenic genes, neutral lipid content
and inflammatory cytokines. …………………………………………………………………....133
Figure 4. Effect of L-GPR120 on neutrophilia and monocytosis. …………………………...134
Figure 5. Effect of L-GPR120 on monocyte recruitment to aortic root intima………………135
Figure 6. Histological quantification of aortic root atherosclerotic lesions…………………..136
Figure 7. Effect of L-GPR120 on aortic cholesterol content. ………………………………..137
Supplementary Table 1: Atherogenic Diet (AD) percentage fatty acid composition (%FA) and
percentage total energy equivalence (% EE) of individual fatty acid……………………….145
Supplementary Figure 1. Bone marrow transplantation (BMT) efficiency. ………………...152
Supplementary Figure 2. Body weight gain and terminal body weights…………………….153
Supplementary Figure 3. Terminal organ/body weight ratios ……………………………….154
Supplementary Figure 4. RBC fatty acid (FA) composition…………………………………..155
Supplementary Figure 5. Liver Histology……………………………………………………….156
Supplementary Figure 6. Effect of L-GPR120 on circulating monocytosis. ………………...157
Supplementary Figure 7. Monocyte recruitment experiment. ………………………………. 158
Supplementary Figure 8. Representative aortic root atherosclerotic lesions
stained for Oil-red-O. …………………………………………………………………………….159
Supplementary Figure 9. Representative aortic root atherosclerotic lesions
stained for CD68/ macrophages. ……………………………………………………………….160
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Supplementary Figure 10. Representative aortic root atherosclerotic lesions
stained for Sirius red/ collagen…………………………………………………………..………161
Supplementary Figure 11. Representative aortic root atherosclerotic lesions
stained for CD11c/ dendritic cells……………………………………………………………….162
Supplementary Figure 12. Representative aortic root atherosclerotic lesions
stained for cleaved-caspase-3/ apoptotic cells………………………………………………..163
CHAPTER IV:
Figure 1. Summary of potential pathways for decreased inflammation
by n-6 and n-3 fatty acids ……………………………………………..…………….…………..175
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LIST OF ABBREVIATIONS
13-HODE 13-hydroxyoctadecadienoic acid
15-HETE 15-hydroxyeicosatetraenoic acid
15d-PGJ2 15-Deoxy-delta 12,14-prostaglandin J2
ABCA1 ATP-binding cassette transporter A1
AP-1 activator protein 1
apoE apolipoprotein E
apoEKO apolipoprotein E knockout
Arg-1 arginase-1
CCR2 C-C chemokine receptor type 2
CCR7 C-C chemokine receptor type 7
CD cluster of differentiation
CHD coronary heart disease
COX cyclooxygenase
CVD cardiovascular disease
CX3CR1: CX3C chemokine receptor 1
DART The Diet and Reinfarction Trial
DGLA dihomo-gamma-linolenic acid
DHA docosahexaenoic acid
EPA eicosapentaenoic acid
GISSI Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto Miocardico
GLA gamma-linolenic acid
Gr-1 granulocyte differentiation antigen 1
HDL high density lipoprotein
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IFN-γ interferon gamma
IL-1 interleukin 1
iNOS inducible nitric oxide synthase
JELIS Japan EPA Lipid Intervention Study
LP lipoprotein
LDL low density lipoprotein
LDLr low density lipoprotein receptor
LDLrKO low density lipoprotein receptor knockout
LO lipoxygenase
LP lipoprotein
LPS lipopolysaccharide
LXR liver X receptor
Ly6C lymphocyte antigen 6 C
Ly6G lymphocyte antigen 6 G
mmLDL minimally modified LDL
MMP matrix metalloproteinase
MR mannose receptor
NCoR nuclear receptor corepressor
NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells
oxLDL oxidized LDL
PIAS1 protein inhibitor of activated STAT1
PPAR peroxisome-proliferator activated receptor
SDA stearidonic acid
SMC smooth muscle cell
STAT signal transducers and activators of transcription
Th T helper lymphocyte
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TNF-α tumor necrosis factor alpha
VCAM-1 vascular cell adhesion molecular 1
VLDL very low density lipoprotein
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ABSTRACT
Swapnil V. Shewale
THE ROLE OF BOTANICAL OILS ENRICHED IN FADS2-DERIVED N-3 VS. N-6 POLYUNSATURATED FATTY ACIDS IN PREVENTION OF ATHEROSCLEROSIS
Dissertation under the direction of
John S. Parks, Ph.D., Professor, Internal Medicine/Section on Molecular Medicine
Background: Dietary polyunsaturated fatty acids (PUFAs) reduce atherosclerosis in animal
models and humans relative to dietary saturated and monounsaturated fatty acids. Although
some of the atheroprotection of dietary PUFAs is due to plasma lipid lowering, in vivo
conversion of 18 carbon PUFAs through the rate-limiting fatty acid desaturase-2 (FADS2,
delta-6 desaturase) step of fatty acid desaturation and elongation results in 18 and ≥ 20
carbon PUFAs that are substrates for pro-inflammatory and anti-inflammatory eicosanoid
production, which affect atherosclerosis progression and inflammation. We previously showed
that an atherogenic diet containing echium oil (EO), which is relatively enriched in stearidonic
acid (18:4 n-3), the immediate product of FADS2-mediated desaturation of 18:3 n-3,
effectively enriches plasma and tissue lipids in the anti-inflammatory PUFA 20:5 n-3 and was
as atheroprotective as dietary fish oil (FO) compared to palm oil (PO), which is enriched in
saturated and monounsaturated fatty acids. However, whether a similar strategy of dietary
enrichment in FADS-2 n-6 products would lead to atheroprotective is unknown. To address
this gap in knowledge, we tested the hypothesis that dietary borage oil (BO), enriched in the
FADS-2 product 18:3 n-6, would not be as atheroprotective as EO, due to in vivo conversion
to 20:4 n-6, a pro-inflammatory eicosanoid precursor. We also investigated the role an anti-
inflammatory protein G-protein coupled receptor 120(GPR120) that is activated by PUFAs in
atheroprotection and hypothesized that dietary n-3 PUFAs would lead to greater activation of
GPR120 and less inflammation than n-6 PUFAs.
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Two studies were performed using: 1) LDL receptor knockout (LDLrKO) mice, or 2) irradiated
LDLrKO mice transplanted with wild type (WT) or GPR120 knockout (KO) bone marrow. Mice
were fed one of four atherogenic diets containing 0.2% cholesterol and 10% fat as PO + an
additional 10% of fat as PO, FO, EO or BO for 8-16 weeks. Measurements of lipid
metabolism, atherosclerosis, and inflammation were made to test out hypotheses.
In study 1, mice fed BO, EO and FO vs. PO had significantly lower plasma total and VLDL
cholesterol concentrations, hepatic neutral lipid content and inflammation, aortic CE content,
aortic root intimal area and macrophage content, and peritoneal macrophage inflammation,
CE content, and ex vivo chemotaxis. We conclude that botanical oils enriched in 18:3 n-6 and
18:4 n-3 PUFAs beyond the rate-limiting FADS2 enzyme are equally effective in preventing
atherosclerosis and hepatosteatosis compared to saturated/monounsaturated fat due to
cellular enrichment of ≥20 PUFAs, reduced plasma VLDL, and attenuated macrophage
inflammation.
In study 2, mice fed BO, EO and FO vs. PO had significantly reduced plasma cholesterol,
triglycerides, VLDL cholesterol, hepatic steatosis, and atherosclerosis that were equivalent for
mice transplanted with WT and GPR120 KO mouse bone marrow, demonstrating that leukocyte
GPR120 expression did not affect these outcomes. In BO, EO and FO, but not PO-fed mice,
lack of leukocyte GPR120 resulted in neutrophilia, pro-inflammatory Ly6Chi monocytosis,
increased monocyte recruitment into aortic roots, and increased hepatic inflammatory gene
expression. We conclude that leukocyte GRP120 expression has minimal effect on dietary
PUFA-induced plasma lipid/lipoprotein reduction and atheroprotection, and that there is no
distinction between n-3 vs. n-6 PUFAs in activating anti-inflammatory effects of leukocyte
GPR120 in vivo.
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CHAPTER I
INTRODUCTION
Swapnil V. Shewale
Swapnil V. Shewale prepared this chapter. Dr. John S. Parks acted in an advisory and editorial
capacity
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1. Cardiovascular diseases and polyunsaturated fatty acids (PUFAs)
1.1 Prevalence of Cardiovascular diseases (CVD) and dietary recommendations: CVD,
including coronary heart disease (CHD) and stroke, is the leading cause of mortality in North
America affecting about one third of adults and was estimated to cost $320 billion dollars in
direct and indirect costs in year 20111. The hallmarks of CVD are elevated plasma cholesterol
and accumulation of lipid-laden macrophage foam cells in the subendothelial space (i.e., intima)
of arteries 2. Intimal monocyte recruitment followed by differentiation into macrophages that
scavenge modified apolipoprotein B containing (apoB) lipoproteins (LP) and apoB LP-
proteoglycan complexes leads to foam cell formation and eventually results in atherosclerosis 2
3. Progressive obstruction of the vessel lumen due to thickening of the intimal area, ulceration,
thrombosis, or embolization of a plaque results in myocardial infarction or stroke2. One of the
first approaches to CVD treatment is to: 1) reduce saturated fat and cholesterol intake, and 2)
replace saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) with n-3
polyunsaturated fatty acids (PUFA) from fish oil (FO) and/or n-6 PUFA from vegetable oils4. As
a set of goals, the AHA recommends consumption of <300 mg cholesterol/day, ~25-35% of total
calories from fat, and up to 10% of total calories from PUFAs 5, 6. Understanding the mechanism
by which different types of dietary fatty acids affect plasma lipid-lipoprotein metabolism,
macrophage function, and atherosclerosis development is important for public health.
2. Dietary n-3 and n-6 PUFAs are atheroprotective
2.1 Epidemiological evidence for atheroprotection by n-3 PUFA: Dyerberg et al in their
landmark observations in the 1970’s noted that death from CHD in Greenland Eskimos was
significantly lower compared to their Danish counterparts. These effects were attributed to their
diets and later on fatty acid analysis of Eskimo diets revealed that, although both Greenlanders
and their Danish counterparts consumed a high fat (~40%) diet, Greenlanders consumed fewer
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SFAs (22% vs. 55%), and increased MUFAs (~57% vs. ~35%) and PUFAs, specially n-3
PUFAs (~13.7% vs. ~2.1%). The reduced CHD events were attributed to anti-thrombotic effects
of n-3 PUFAs, especially eicosapentaenoic acid (EPA, C20:5 n-3) and docosahexaenoic acid
(DHA, C22:6 n-3) obtained from fatty fish 7, 8. Other pleotropic benefits have been attributed to
diets enriched in fatty fish, including plasma TG lowering and reduced inflammation to name a
few 6, 9, 10. Since then, atheroprotective effects of replacing dietary SFAs and MUFAs by n-3
PUFA containing diets has been demonstrated in humans and in several animal models,
including African green monkeys and transgenic mice 11-16. For example, in a primary endpoint
prevention trial named Effects of eicosapentaenoic acid on major coronary events in
hypercholesterolemic patients (JELIS), EPA consumption (1800 mg/day, 5 years) resulted in a
19% relative reduction in major coronary events (primary endpoint) in hypercholesterolemic
subjects17. Other secondary endpoint prevention trials including, The Diet and Reinfarction Trial
(DART) 18, GISSI-Prevention Study19, Nurses’ Health Study20 and Physicians’ Health21 Study
have shown a protective effects of FO consumption with regard to risk and/or outcomes for CVD
including CHD and myocardial infarction.
2.2 Epidemiological evidence for atheroprotection by n-6 PUFA: Although much attention
has been given to n-3 PUFAs, n-6 PUFAs are also atheroprotective in mouse models of
atherosclerosis, non-human primates, and in human populations22-25. Some of the
atheroprotection is likely due to plasma lipid lowering, but there may also be an anti-
inflammatory role for n-6 PUFAs through the generation of prostaglandin E1 (PGE1) from
dihomo-γ-linolenic acid (DGLA, 20:3 n-6), which is a potent inhibitor of thromboxane A2 (TXA2)
formation and inhibits leukocyte adherence to endothelial cells26, 27. In addition, a recent meta-
analysis of 13 cohort studies involving 310,602 individuals and 12,479 coronary heart disease
events revealed an inverse association between dietary linoleic acid (LA, 18:2 n-6) intake and
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coronary heart disease risk, such that, a 5% increase in energy intake from LA was associated
with a 10% and 13% lower risk of coronary heart disease events and deaths, respectively 28.
3. Overview of lipid and lipoprotein metabolism
3.1. Circulating lipids and lipoproteins: Lipids in circulation are transported as hydrophilic,
spherical structures called lipoproteins (LP). LP are composed of: a) amphipathic
apolipoproteins on the surface that assist in providing a spherical structure to LP and can act as
cofactors and ligands for lipid-processing enzymes, b) a neutral lipid core, consisting of
triglycerides (TG) and cholesteryl esters (CE), and c) a monolayer of polar lipid, consisting of
free cholesterol (FC) and phospholipids (PL), mainly phosphatidylcholine (PC) and
sphingomyelin lipids29. Circulating lipoproteins can be categorized based on a specific
apolipoprotein associated with the given LP, their electrophoretic mobility, size and/or density
(ratio of lipid to protein) as outlined in Table 1 30.
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Table 1. General classification of circulating lipoproteins
Classification
Scheme
Chylomicron VLDL LDL HDL
Density (g/ml)
<0.93 0.95-1.006 1.019-1.063 1.063-1.21
Size (nm) 75-1200 30-80 18-25 7-12
Major cargo Triglyceride Triglyceride Cholesterol Cholesterol,
Phospholipid
Apolipoprotein/s ApoB48, ApoCII,
ApoCIII,
ApoE, ApoAIV
ApoAI, ApoAII
ApoB100, ApoE,
ApoCI, ApoCII,
ApoCIII
ApoB100,
ApoCII,
ApoCIII, ApoE,
Apo(a)
ApoA I,
ApoAII,
ApoCII, ApoCIII,
ApoE
Electrophoretic
mobility
Origin/ α2 Pre-β β α
Apo, apolipoprotein.
Intermediate density lipoproteins (IDL, d=1.006-1.019 g/ml) are VLDL remnants resulting from
VLDL TG hydrolysis by lipoprotein lipase (LPL) and are usually not detectable in the circulation
due to rapid removal from plasma or conversion to LDL31.
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3.2. Overview of lipoprotein metabolism
3.2.1. Chylomicrons: Approximately 95% of dietary fat is TG and the remainder is free fatty
acid (FFA), PL, CE and fat soluble vitamins. In the small intestinal lumen, several lipases,
including carboxyl ester lipase / cholesterol esterase, intestinal pancreatic triglyceride lipase and
group 1B phospholipase A2, hydrolyze lipids into absorbable FC, lysophospholipids, glycerol
and FFAs 32, 33. Digested lipids are then solubilized by bile acids into micelles for absorption into
intestinal epithelial cells (i.e., enterocytes)34. Once absorbed into enterocytes, FFAs are re-
esterified into TG35, FC is esterified to CE by sterol O-acyltransferase 2 (SOAT2, also known as
acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT2))36, 37 and packaged with apoB-48 into
chylomicrons (CM), the largest lipoprotein. ApoB-48 consists of the amino terminal 2152 amino
acids of apoB-100, resulting from a unique APOBEC-1 mRNA-editing mechanism38. In humans,
apoB-48 is exclusively synthesized in intestine, whereas human liver only produces only apoB-
100 39. Chylomicrons, due their large size (75-1,200 nm) (Table 1), enter the circulation through
the lymphatic system40. Once chylomicrons are released in the circulation, lipoprotein lipase
(LPL) hydrolyses TG in chylomicrons, releasing glycerol and FFA41 for energy storage (adipose
tissue) or utilization (muscle) (Figure 1, Table 2)).
3.2.2. Very low density lipoprotein (VLDL): VLDL is a liver derived LP enriched in TG (Table
1) and functions to distribute FFFAs to different tissues through hydrolysis of TG by LPL. In a
fasting state, intracellular TG and CE is mobilized (hydrolyzed and re-esterified) from lipid
droplets, assembled with apoB-100 and secreted as VLDL particles. In the ER, prior to
incorporation in VLDL, FC is esterified to CE via SOAT2 36, whereas, FFA are esterified to TG
and PL in the sn-glycerol-3-phosphate pathway via sequential reactions involving acyl-
transferases42. Assembly of apoB-100 into VLDL involves sequential steps where an ER-
localized cofactor, microsomal triglyceride transfer protein directs the cotranslational lipidation of
apoB-100 in the rough ER membrane with phospholipid and TG to form precursor VLDL
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particles with diameters of ∼10–20 nm. A second step of particle assembly involves movement
of cytosolic lipid into the ER to form luminal lipid droplets, which then fuse with precursor VLDL
particles, forming mature VLDL that are secreted in circulation 43, 44.
3.2.3. Low density lipoprotein (LDL): Gradual VLDL TG lipolysis gives rise to IDL and
ultimately to LDL. LDL is the major plasma cholesteryl ester-transporting lipoprotein (Table 1).
LDL is catabolized via binding to the LDL receptor (LDLr) on the cell surface of the liver and
extrahepatic tissues45 (Figure 1).
3.2.4. Intravascular metabolism of apoB containing LP by LPL:Several genes have been
identified whose loss of function results in severe plasma hypertriglyceridemia. These include
LPL, APOC2, APOA5, lipase maturation factor 1, and glycoprotein glycosylphosphatidylinositol
(GPI)-anchored high-density lipoprotein (HDL)-binding protein 1 (GPIHBP1)46. LPL is bound to
heparin sulphate proteoglycans on endothelial cell surface in peripheral tissues. Once
chylomicrons and VLDLs are released in the circulation, LPL hydrolyse TG, releasing glycerol
and FFA41 for energy storage (adipose tissue) or utilization (muscle) (Figure 1). APOC2 and
APOA5, are known to stimulate LPL activity, whereas APOC1 and APOC3 inhibit LPL activity.
GPIHBP-1 is known to bind both chylomicrons and LPL and is thought to act as an anchoring
protein that stabilizes LPL. Additionally, GPIHBP-1 can interact with angiopoietin-like protein 4
(Angptl4), which is known to inhibit LPL activity47. After chylomicron TG lipolysis, the residual
CE-rich, TG-depleted lipoprotein particles, referred to as chylomicron remnants, are released
into the plasma compartment and rapidly cleared by the liver through chylomicron remnant
receptors, LDLr, and LDL-receptor related protein 48, 49.
3.2.6. High density lipoprotein (HDL): HDL biogenesis involves the synthesis and secretion of
the major HDL-associated apolipoproteins, apoA-I, synthesized in liver and intestine, and apoA-
II, synthesize in liver. Following secretion, apoA-I and apoA-II are lipidated via ATP binding
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cassette transporter A1 (ABCA1) at the cell membrane surface, resulting in nascent HDL
(nHDL) particle formation. Subsequent maturation of nHDL to spherical plasma HDL occurs in
the circulation50.
3.2.7. Intravascular HDL maturation and reverse cholesterol transport: Maturation of nHDL
involves acquisition of additional PL from redundant VLDL surface generated during LPL
lipolysis of TG, and PL transfer from other plasma apoB LP via phospholipid transfer protein
(PLTP) (Figure 1)51. Maturation of HDL in the circulation also involves esterification of FC to CE
via lecithin: cholesterol acyltransferase (LCAT), generating a neutral lipid core (Figure 1)52.
Liver is the largest contributor to the plasma HDL pool 53. CE-enriched HDL returns to the liver
in a process known as reverse cholesterol transport (RCT), which is the only quantitative way of
removing extra-hepatic cholesterol from body54, 55. Liver is the primary site of HDL cholesterol
uptake from plasma via scavenger receptor class BI (SR-BI)-mediated selective HDL CE uptake
without HDL apolipoprotein degradation56, 57. Another circulating protein, CE transfer protein
(CETP), plays a critical role in human HDL cholesterol catabolism. CETP exchanges TG from
apoB LP for CE in HDL, leading to TG enrichment and CE depletion in HDL particles.
Individuals with loss of function mutations in the CETP gene have high HDL cholesterol levels,
whereas in rodents, which lack CETP, overexpression of CETP results in significant reductions
in HDL-C58-60 (also reviewed in Figure 1, Table 2).
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Figure 1. Outline of lipoprotein transport and metabolism
(adapted from Michael R. Flock et al. Adv Nutr 2011;2:261-274)
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Table 2. Major enzymes important in lipoprotein metabolism
Enzyme /
transporter
Location Function
ABCA1 Cell membrane Free cholesterol and phospholipid efflux
CETP Circulation/HDL Cholesteryl ester (CE) transfer from VLDL to HDL and
TG transfer from HDL to VLDL
LPL Endothelium Catalyzes chylomicron and VLDL TG hydrolysis to
release FFA. Activated by apoC-II and inhibited by
apoC-III
LCAT Circulation/HDL Esterification of free cholesterol to CE to generate
mature HDL particles. Activated by apoA-I and other
apolipoproteins
PLTP Circulation Phospholipid transfer from VLDL/LDL to HDL
SOAT2 Liver and
Intestine
Generation of intracellular CE from esterification of FC.
CE can be stored in lipid droplets or secreted in VLDL
particles
ABCA1: ATP binding cassette transporter 1, CETP: cholesteryl ester transfer protein, LPL:
lipoprotein lipase, LCAT: lecithin cholesteryl acetyl transferase, PLTP: phospholipid transfer
protein, SOAT2: sterol O-acyltransferase 2. (Note: Above enzymes are also depicted in
Figure. 1).
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4. PUFA-induced transcriptional regulation of lipoprotein metabolism and inflammation
PUFAs, in addition to providing energy through β-oxidation, modulate cellular functions in
multiple ways including regulation of membrane composition and fluidity, cell signaling (i.e.
phosphorylation, ubiquitination or proteolytic cleavage), and binding to transcription factors
and/or their co-activators61. Nuclear receptors (NR) consist of a DNA-binding domain (DBD) and
a ligand-binding domain (LBD). Ligand binding induces a conformational change that results in
NR binding to DNA (as homo/heterodimers) or to DNA-bound transcription factors. NR
themselves can act as co-activators or co-repressors by binding to other transcription factors
present in regulatory regions of target genes (reviewed in 62).
4.1. Nuclear receptors that directly bind to unsaturated fatty acids:
Fatty acids share the property of hydrophobicity with hormones (e.g. steroid, thyroid hormones)
that activate NR. Several NR including peroxisome proliferator receptor (PPAR: α, β, γ1, γ2
isoforms), hepatocyte nuclear factor-5 (HNF4), retinoic acid receptor (RXR: α) and liver X
receptor (LXR: α and β isoforms) contain fatty acid binding domains. Although, fatty acid
(especially linoleic acid) binding (reversible as well as irreversible) to HNF4 has been
documented, its effects on transcriptional activity and physiological implications are relatively
underexplored.
4.1.1: PPARs: Via their DBD, PPARs bind to peroxisome proliferator response elements
(PPREs) in enhancer region of target genes as heterodimers of RXR 63. Among three known
human PPAR isotypes, α, γ and δ, PPARγ and PPAR δ are abundantly expressed in the
macrophage64. PPARα is predominantly expressed in (rodent and human) liver and brown
adipose tissue, and increases expression of genes involved in fatty acid elongation,
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desaturation, oxidation and transport to accommodate increased requirement for fatty acid
oxidation upon fasting 65. Macrophage PPARα also has atheroprotective potential 66.
PPARγ is unique in having a large T-shaped cavity of the apparent volume (~1,300 A˚) as a
ligand binding site for rosiglitazone and naturally occuring hydrophobic carboxylic acids,
including essential fatty acids and their oxidized derivatives (such as linoleic acid derived 13-
HODE, 15-HETE), and prostaglandin J2 metabolites, such as (15d-PGJ2) 67-69. Longer chain
PUFAs typically bind to PPARγ in the micromolar concentration range 68 and induce
“transrepression” of NF-κB activated inflammatory genes70, 71.
Macrophage PPARγ is implicated in differentiation, ox-LDL uptake, and foam cell formation67, 72.
PPARγ is also expressed in human atherosclerotic plaque macrophages 73 and its deletion in
mice exacerbates atherosclerosis compared to their WT counterparts given synthetic PPARγ
agonist 74. Additionally, n-3 PUFAs share anti-inflammatory, insulin-sensitizing, and triglyceride-
lowering effects with PPAR agonists, thiazolidinediones (TZDs) and fibrates, which target
PPARγ and PPARα, respectively (reviewed in 75).
4.1.2. LXRs: LXRs (α and β isotypes) are central regulators of lipid homeostasis. LXRα is
expressed primarily in liver, intestine, adipose tissue, and macrophages, whereas LXRβ is
expressed in many cell types76. In peripheral cells, such as macrophages, LXRs directly control
transcription of genes involved in cholesterol efflux pathways, including ABCA1, ABCG1 and
apoE77-79. In the intestine, ligand activation of LXR/RXR heterodimers dramatically reduces
dietary cholesterol absorption, an effect postulated to be mediated by upregulation of LXR target
genes ABCA1 and ATP binding casette sterol transporters, ABCG5/G8 80, 81. In the liver, LXRs
regulate cholesterol and fatty acid metabolism and bile acid synthesis82. Additional work has
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focused on identifying unique functions for LXRα and β isoforms and on synthetic antagonists
that could be used to treat atherosclerosis and metabolic disorders (reviewed in83-85).
In vitro, unsaturated fatty acids prevent LXRs binding to its coactivators86, resulting in lower
transcription of LXR target genes, including SREBP1-c, even in presence of synthetic or
endogeneous LXR ligands, such as 24(S),25-epoxycholesterol and 22(R)-hydroxycholesterol87.
Additionally, PUFAs induce PPARγ activation, which inhibits LXR-RXR heterodimer formation
and reduces LXR activation88.
4.2. Regulation of transcription factors by unsaturated fatty acids without direct binding
4.2.1. Sterol Regulatory Element-Binding Proteins (SREBPs): SREBPs belong to the basic
helix-loop-helix-leucine zipper (bHLH-Zip) family of transcription factors and include SREBP-1a,
SREPB-1c and SREBP2. SREBP-1a and -1c are “splice variants” produced from a common
gene. SREBP-1c is predominantly expressed in liver and is selective for the fatty acid pathway,
whereas SREBP-1a is expressed in all other cells and is a strong activator of cholesterol and
fatty acid biosynthesis. SREBP-2 is encoded by a separate gene and is selective for cholesterol
biosynthesis in liver 89.
4.2.2. Negative feedback regulation of de novo lipogenesis via SREBPs: SREBPs are
synthesized as inactive ER membrane protein precursors (consisting of 1150 amino acids).
SREBP cleavage-activating protein (Scap) is a tetrameric ER membrane protein that binds both
sterol and SREBPs. ER cholesterol content is estimated to be ~1 molar percent of total cellular
cholesterol and when ER membrane cholesterol content falls below ~5 mole percent with
respect to phospholipid, Scap acts as an escort protein by binding to COPII vesicular proteins
and mediating ER to Golgi translocation of SREBP2 90. In the Golgi, two proteases (site 1 and
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site 2 protease, S1P and S2P, respectively) cleave off the N-terminus of SREBPs to release a
transcriptionally active basic helix-loop-helix leucine zipper fragment, which in the nucleus
activates lipogenic genes. When ER cholesterol content rises above ~5 mole percent, SCAP
undergoes a conformational change that results in its binding to Insulin-induced gene protein
(INSIG). Upon Scap-Insig binding, COPII complex can no longer bind to Scap, hence retaining it
in the ER. Inhibition of ER-Golgi translocation of the SREBP/Scap complex results in SREBP
feedback inhibition (reviewed in90, 91).
4.2.3. Carbohydrate/glucose responsive transcription factor (ChREBP): In liver, SREBP-1c
overexpression induces hepatosteatosis 92, whereas its deletion does not completely shut down
fatty acid synthesis (~50% reduction) 93. This was postulated to be likely due to the presence of
Glucose-or-carbohydrate-response elements (GhoREs) in promoters of many lipogenic genes94.
Since, GhoREs can be recognized by bHLH family members (including SREBPs) 95, a large
protein (864 amino acids) containing a bHLH domain was discovered and identified as
ChREBP96. ChREBP and SREPB-1c activation induces glycolytic and lipogenic gene
expression in a synergistic fashion and in a gluckokinase-dependent manner97, whereas
ChREBP deletion reduces glycolysis and lipolysis, resulting in decreased hepatic steatosis,
insulin resistance, and increased β-oxidation in mice98, 99. PUFAs (in vitro as well as in vivo)
inhibit ChREBP activity by decreasing its mRNA stability and its translocation from the cytosol to
the nucleus100.
4.2.4. Nuclear factor kappa B (NF-κB): NF-kB regulates expression of the genes involved in
innate and adaptive immune responses. Activation of canonical NF-kB pathway up-regulates
genes encoding cytokines, adhesion molecules anti-apoptotic and proliferative pathways. NF-kB
exists in the form of a cytosolic trimer consisting of RelA or RelB, p50 or p52, and an inhibitory
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protein, IκB. NF-kB activation by extracellular signals, such as endotoxins, cytokines, free
radicals, results in phosphorylation, dissociation, and degradation of IkB, releasing the NF-kB
dimer to translocate to the nucleus where it upregulates pro-inflammatory target gene
expression 101.
In vitro, EPA and DHA inhibit of inflammatory proteins in endothelial cells, monocytes,
macrophages and dendritic cells. The inhibitory effects of n-3 PUFA involve decreased IkB
phosphorylation resulting in inactivation of NF-kB 102, and inactivation of key early signaling
proteins, such as mitogen-activated protein kinases103. Dietary supplementation of n-3 PUFA in
animals decreases NF-kB activation104, whereas in humans, 10 weeks of FO feeding
(18gm/day) reduced IL-1β and TNF-α secretion from endotoxin-stimulated mononuclear cells105.
Additional evidence suggests that ligand bound (i.e., activated) PPARs physically interact with
NF-kB, preventing its translocation to the nucleus106.
5. Immunomodulation by n-3 and n-6 PUFAs and their oxidized derivatives via G-protein
coupled receptors (GPCR)
5.1. Eicosanoid generation via n-3 and n-6 PUFA: Arachidonic acid (AA, 20:4 n-6) esterified at
the sn-2 position in cellular phospholipids (PLs) is a precursor for generation of oxidized
metabolites, collectively called eicosanoids, via the cyclooxygenase (COX) and lipoxygenase (LO)
pathways, resulting in families of inflammatory mediators, such as leukotrienes (LTs),
prostaglandins (PGs), and thromboxanes (TXs)107. These mediators are rapidly synthesized and
released from cells in response to extracellular stimuli. They act on cells in an autocrine or
paracrine fashion through GPCRs to initiate downstream second messengers that modulate
inflammation, chemotaxis, and vascular function12. Conversely, EPA, an n-3 counterpart of AA,
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can generate oxidized metabolites that include series 3 PGs and TXs, series 5 LTs, and hydroxy-
eicosatetraenoic acids (HETEs) by the same pathways, resulting in n-3 eicosanoid products that
are typically less active than their n-6 counterpart in eliciting an inflammatory response. For
instance, leukotriene B5 (LTB5), a major end product of EPA catalysis by 5-LO, has 10% of the
biological activity of LTB4, the analogous end product of 5-LO from AA12, 108. Additionally, EPA can
replace AA in the PL fraction of the cell and compete with AA for the pathways involved in
eicosanoid production, leading to reduced series 2 PGs and TXs, and series 4 LTs and HETEs108.
5.2. Free fatty acid receptors (FFARs): Recent deorphanization of FFARs has allowed further
understanding of FFAs as signaling molecules. FFA receptors are G-protein coupled receptors
(GPRs) that are activated by short, medium or long chain FFAs109-114 and include GPR40
(FFAR1), GPR43 (FFAR2), GPR41 (FFAR3), GPR84 and GPR120 (FFAR4). GPR120 is highly
expressed in intestine, adrenals, lung, adipose tissue, and macrophages and is described as an
n-3 PUFA receptor115. Upon activation by n-3 PUFA, GPR120 inhibits transforming growth factor
beta-activated kinase 1 (TAK1) activation, resulting in attenuation of NF-κB and Jun N-terminal
kinases (JNK) signaling115. GPR120 regulates glucose metabolism and obesity in mice and
humans; a non-synonymous mutation (p.R270H) inhibited GPR120 signaling activity, resulting
in increased risk of obesity in European populations116.
Although macrophages, adipose tissue, and gut predominantly express GPR120, the relative
contribution of its expression in various cell types is just beginning to be determined. Bone
marrow transplantation studies by Oh et al demonstrate that the insulin-resistance and pro-
inflammatory macrophage phenotypes observed in whole body GPR120 KO mice can largely be
attributed to hematopoietic cell/ macrophage GPR120 deletion. In LPS primed macrophages,
GPR120 activation by DHA or a synthetic agonist results in its internalization via βarr2 and
binding to TAB1, preventing TAB1 and TAK1 kinases interaction required to activate pro-
inflammatory NF-κB and JNK pathways 115, 117. Downstream of GPR120 and GPR40, βarr2-
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TAB-1 interaction also prevents βarr2 and NLRP3/NLRP1c binding required for inflammasome
activation, reducing caspase-1 activation and IL-1β maturation 118. GPR120 activation by DHA
attenuates LPS-induced macrophage COX2 induction, PGE2 production, and IL-6 gene
expression119. In mouse liver, GPR120 expression is localized to Kupffer cells and its activation
protects mouse livers from ischemia reperfusion injury.
6. Macrophages and monocytes in atherosclerosis
6.1. Macrophage activation and polarization: Macrophages are professional phagocytotic
cells of the innate immune system120 that recognize pathogen-associated molecular patterns
(PAMPs) via expression of the toll like receptors (TLRs)121, 122. Downstream signaling events of
TLR activation are mediated by association of intracellular adaptor proteins with TLR signaling
domains. TLR-4 is activated by lipopolysaccharide (LPS) (a cell wall component of gram
negative bacteria) and signals through its co-receptor, CD14. The TLR-4-mediated signaling
pathway is well characterized and known to activate NF-κB and MAP kinases.
Tissue resident macrophages can be “polarized” toward a pro-inflammatory M1 or anti-
inflammatory M2 phenotype123, 124. Activation by Th1 stimuli (e.g. IFN-γ, LPS) induces iNOS
synthase required for intracellular pathogen killing and results in secretion of proinflammatory
cytokines, such as TNF-α, IL-1β, IL-6, IL-18, and IL-12p40. Activation by Th2 stimuli (e.g. IL-4,
IL-13) derived from various cells of the innate immune system125, as well as adipocytes 126,
induces expression of the mannose receptor (i.e. CD206)127 and arginase-1. M2 macrophages
secrete anti-inflammatory cytokines, such as IL-10, and TGF-β, and are implicated in resolution
of inflammation and wound healing 124, 128.
TLRs can also be aseptically activated via SFAs and endotoxins. FetA, a liver-derived
circulating glycoprotein, is an adaptor protein that can act as a carrier of SFAs resulting in TLR4
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activation22, 23. Additionally, n-3 PUFAs can limit TLR4 dimerization to membrane lipid rafts and
inactivate the NF-κB pathway129. Deletion of MyD88, an adaptor protein downstream of TLR4-
CD14 complex, results in reduced atherosclerotic plaque and circulating levels of monocyte
chemoattractant protein-1 (MCP-1)130, whereas deletion of TLR4, but not CD14, also reduces
atherosclerosis and is accompanied by reduced circulating levels of MCP-1131.
6.2. Monocyte Subsets and monocyte recruitment to atherosclerotic lesions: Like
macrophages, monocytes differ in their effector function and can be classified so. Circulating,
undifferentiated monocytes respond to their environment and chemotactic cues by recruitment
and differentiation into macrophages and/or dendritic cells. In mice, two major monocyte
populations have been described based on cell surface C-C chemokine receptor (CCR) and
CX3C chemokine receptor (CX3CR) expression as: 1) CCR2+, CX3CR1+, Ly-6Chi (i.e., Ly-6Chi) or
pro-inflammatory monocytes and 2) CCR2-, CX3CR1++,Ly-6Clo (i.e. Ly-6Clo) or patrolling
monocytes 132, 133. CCR2 is the receptor for MCP1, which has been shown to be important in
attracting blood monocytes to developing atherosclerotic lesions25. Ly6Chi monocytes are
phenotypic correspondents to human CD16-CD14+ monocytes134. Nonclassical, Ly6Clo monocytes
have been termed resident/reparative in mice because of their longer half-life in vivo; they are
thought to replenish tissue-resident dendritic cells and macrophages. Ly6Clo monocytes are
phenotypic correspondents to CD16+ CD14dim human monocytes134, 135.
6.3. Hypercholesterolemia-induced monocytosis: Hypercholesterolemia is associated with
increased monocytes in the circulation that display a more inflammatory phenotype associated
with greater surface expression of Ly6Chi and CCR2, the receptor for the chemokine CCL2 (i.e.,
MCP1) that is involved in monocyte recruitment to sites of inflammation 25, 33. A greater proportion
of these monocytes are found in atherosclerotic lesions as macrophages, suggesting that these
monocytes are preferentially recruited to developing atherosclerotic lesions34, 35. The
accumulation/influx of blood monocytes in the artery wall correlates with lesion development and
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occurs throughout the progression of atherosclerosis136. Deficiency CCR2 or CX3CR1 or their
ligands inhibits recruitment of specific monocyte subsets to the artery wall137, 138. Furthermore, in
models of atherosclerotic regression, particularly after reversal of hyperlipidemia, monocyte-
derived cells emigrate out of plaques139-141, and monocyte recruitment is also attenuated142.
During regression, monocyte-derived cells take on dendritic cell-like characteristics and employ
CCR7 to exit lesions143.
7. Mouse models of atherosclerosis: C57BL/6 mice do not develop atherosclerosis when fed
a chow diet; however feeding a diet containing 15% fat, 1.25% cholesterol, and 0.5% cholic acid
(Paigen diet)144 results in small lesions in the aortic root region of the heart, with minimal
cholesterol accumulation in the aorta145. Since the advent of gene targeting, two genetically-
modified mouse models have been widely used for atherosclerosis research: 1) mice lacking
apolipoprotein E (ApoEKO)146 and 2) mice lacking low density lipoprotein receptor (LDLrKO)147,
both characterized by elevated plasma cholesterol concentrations (Table 3).
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Table: 3. Mouse models of Athero-progression
Mouse
Model Features / Characteristics Causative factor
Baseline
plasma
cholesterol
ApoEKO mice
Spontaneous development of complex
plaques when mice are fed a chow diet;
acceleration of plaque formation when
mice are fed a Western/high fat diet
Accumulation of
cholesterol rich
remnant lipoprotein
particles
~500mg/dl
LDLrKO mice
Development of plaques following
feeding mice a cholesterol and fat-
enriched diet; lipoprotein profile is similar
to that of humans
Delayed clearance of
VLDL and LDL ~250mg/dl
Adapted from: Nature Reviews Immunology 13,709–721(2013) doi:10.1038/nri3520
8. Pathophysiology of atherosclerotic lesion development: Atherosclerosis is a
characterized by dyslipidemia and inflammation. Multiple cell types including endothelial,
smooth muscle, and immune cells play critical roles in atherosclerosis progression10.
Macrophages are involved in lipid metabolism as well as innate immunity 11. Cholesteryl ester
(CE)-loaded macrophages (i.e., foam cells) are a hallmark of atherosclerotic lesions2. Since LDL
is a major transporters of CE in plasma and elevated plasma apoB-100/apoA-1 ratio is an
independent risk factor for CVD148, atherosclerosis is initiated when plasma LDL is elevated.
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LDL crosses the endothelial monolayer by transcytosis and gets trapped and retained in the
arterial intima through binding to electronegative proteoglycans149. LDL retention is followed by
lipoprotein aggregation and modification via enzymatic (e.g. 12/15 lipooxygenase (12-15 LO),
secretory phospholipase 2 (sPLA2), myeloperoxidases (MPO) etc.) and non-enzymatic (e.g.
free radicals, reactive oxygen species etc.) oxidation, primarily in the phospholipid (PL) fraction,
resulting in minimally-modified or oxidized LDL (mmLDL/oxLDL)150-152.
The mmLDL/oxLDLs stimulate endothelial cells to produce chemokines (MCP-1, interferon- γ
(IFN-γ)), adhesion molecules (e.g. intracellular adhesion molecule (ICAM/ CD54), vascular cell
adhesion protein-1 (VCAM-1/CD106), P and E selectins) and growth factors, such as
macrophage colony-stimulating factor (M-CSF) and granulocyte macrophage colony stimulating
factor (GM-CSF), resulting in chemo-attraction, rolling, and infiltration of monocytes into the
lesional intima, where recruited monocytes differentiate into macrophages2, 153-157. MCP-1/CCR2
interaction plays a critical role in monocyte recruitment in atherosclerosis158. mmLDL/oxLDL is
rapidly taken up via scavenger receptors, SR-A and CD36, expressed on activated
macrophages, leading to foam cell formation159-162. The expression of scavenger receptors is
further regulated by PPARγ and pro-inflammatory cytokines, such as TNF-α and IFN-γ72.
Accumulation of cholesterol and oxygenated derivatives of cholesterol results in downregulation
of LDL receptor45, and upregulation of LXR, resulting in the transcriptional activation of a
program of genes that function to efflux the excess cholesterol from the cell78, 79, 83, 163.
Cholesterol efflux to lipid-free apolipoproteins (i.e., apoA-I, apoE) occurs via ABCA1, whereas
efflux to HDL particles occurs via ABCG1164, 165. Unabated sterol accumulation can ultimately
result in apoptotic and/or necrotic cell death and fibrous plaque formation.
In addition to macrophages, smooth muscle cells (SMCs), T cells, and B cells play a role in
atherosclerotic lesions. SMCs proliferation results in fibrous cap formation. In fibrous plaques,
interaction of CD40 with its ligand CD40L (CD154) (expressed on T cells, B cells, macrophages,
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ECs, and SMCs) results in production of inflammatory cytokines, matrix-degrading proteases,
and adhesion molecules. Activated T cells produce proatherogenic cytokines, such as IFN-γ,
which inhibit extracellular matrix production by SMCs, whereas macrophage secreted interstitial
collagenases, gelatinases, and stromolysin that degrade extracellular matrix 166-168 169-171. In
advanced lesions, calcification and neovascularization (growth of small vessels from media)
both influence stability of atherosclerotic lesions2. Intimal calcification occurs due to secretion of
matrix scaffold by specialized cells of osteoblastic potential in response to cytokines and
oxysterols172. The thrombogenicity of lesion depends on tissue factor, a key protein in
coagulation cascade initiation. Production of tissue factor by ECs and macrophages is
enhanced by oxLDL, infection or CD40-CD40L interaction2, 173, 174. Ultimately, if this pathologic
process is not halted or reversed, atherosclerotic plaque rupture occurs, resulting in myocardial
infarction or stroke.
9. Potential of botanically-derived FADS2 n-6 and n-3 PUFAs as a FO substitute for CVD
prevention: PUFAs have pleotropic functions; n-3 PUFAs are cardioprotective and anti-
inflammatory molecules, whereas n-6 PUFAs have cardioprotective and immunomodulatory
properties.
Reduction of dietary cholesterol and total fat intake and a replacement of dietary saturated fatty
acids by PUFAs is still a primary intervention for CVD4. Despite known cardioprotective and anti-
inflammatory properties of FO, consumption of fatty fish or FO supplements is low in the USA175.
Both n-3 and n-6 PUFAs are atheroprotective in mice, non-human primates, and humans11, 12, 15,
16, 20, 22, 23, 28, 176, 177. Despite high consumption of LA and alpha linoleic acid (ALA, 18:3 n-3)
(mostly consumed in vegetable oils), tissue enrichment of their longer chain bioactive products,
AA and EPA, respectively, is limited due to inefficiency of the rate-limiting FADS2 enzyme in the
fatty acid desaturation and elongation pathway(depicted in Figure 2)178. Botanical oils that are
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enriched in ALA do not offer the plasma lipid lowering and atheroprotective properties that are
observed with FO179. Similarly, most sources of botanical n-6 PUFAs are highly enriched in LA,
constituting ~85-90% of the North American dietary PUFA intake 22, 28, which is inefficiently
converted by FADS2 to longer chain PUFAs (i.e, AA) that are precursors of bioactive eicosanoid
species.
To circumvent the inefficient conversion of LA and ALA to longer chain PUFAs, we have
identified botanical oils enriched in n-3 (echium oil, EO; enriched in 18:4 n-3) or n-6 (borage oil,
BO; enriched in 18:3 n-6 (GLA)) PUFAs that are the immediate products of FADS2 desaturation
of ALA and LA, respectively (Figure 2).. We previously showed that EO effectively enriches
plasma and tissue lipids in EPA 180. In LDLrKO mice, isocaloric replacement of palm oil (PO)
with EO attenuated atherosclerosis severity, splenic monocytosis, monocyte influx into aortic
intima, and aortic root intimal macrophage content to an equivalent extent as FO, lending proof
of principle for this strategy16. However, whether a similar strategy will be atheroprotective for
the n-6 PUFA pathway is unknown. GLA can be elongated to DGLA, a precursor of the anti-
inflammatory PGE1, or AA, a substrate for proinflammatory eicosanoid species. No
atherosclerosis studies have been performed with BO and little is known little about its effect on
plasma lipids and lipoproteins.
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Figure 2. Outline of metabolic pathways for PUFA adapted from Calder and Grimble, 2002.
EOBO
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10. Statement of Research Intent
The purpose of this project was to determine the predominant mechanism (i.e. effect on lipid-
lipoproteins vs. immunomodulatory/ anti-inflammatory) by which FADS2 derived n-6 PUFAs vs.
n-3 PUFAs affect atherosclerosis in LDLrKO mice. We hypothesized that a botanical oil
enriched in the FADS2 product 18:3 n-6 (i.e. BO) will not be as atheroprotective as one enriched
in the FADS2 product 18:4 n-3 (i.e. EO) (Specific Aim 1). Contrary to our hypothesis, we
conclude that BO is as atheroprotective and hepatoprotective as EO and FO relative to Palm Oil
(PO) via VLDL-cholesterol lowering and attenuation of macrophage inflammation and
chemotaxis in vitro.
We then determined the relative contribution of G-coupled protein receptor (GPR) 120, to
FADS2-derived n-3 and n-6 PUFA-induced atheroprotection in LDLrKO mice. GPR120 acts as a
negative regulator of macrophage inflammation and chemotaxis upon activation by n-3 PUFA.
Based on available information, we hypothesized that atheroprotection in mice fed n-3 PUFA-
enriched EO or FO is due to a GPR120 (n-3 fatty acid receptor)-mediated switch of pro-
inflammatory M1 macrophages to anti-inflammatory M2 macrophages, whereas
atheroprotection associated with n-6 PUFA-enriched BO is independent of GPR120 (Specific
Aim 2). We transplanted bone marrow (BM) from wild type (WT) and GPR120 knockout
(GPR120KO) mice into lethally-irradiated LDLrKO recipient mice and investigated lipid
metabolism, inflammatory response and atherosclerosis. We conclude that, although in vivo
activation of L-GPR120 attenuates Kupffer cell inflammation, neutrophilia, splenic monocytosis,
and monocyte recruitment into aortic intima, BO, EO and FO-induced atheroprotective is
independent of leukocyte GPR120 activation.
In summary, atheroprotection in n-6 as well as n-3 PUFA (i.e. BO vs. EO and FO) fed LDLrKO
mice is primarily driven by VLDL-cholesterol lowering.
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This thesis addressed the considerable gaps in knowledge regarding atheroprotective and anti-
inflammatory potential of BO supplementation. This work helped elucidate the role of FADS2
derived n-6 fatty acids in atheroprotection. Furthermore, this work elucidated the relative
contribution of a macrophage anti-inflammatory protein GPR120 on atherosclerosis in the
context of n-6 as well as n-3 PUFA feeding.
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CHAPTER II
BOTANICAL OILS ENRICHED IN N-6 AND N-3 FATTY ACID PRODUCTS OF FADS2 ARE
EQUALLY EFFECTIVE IN PREVENTING ATHEROSCLEROSIS AND HEPATOSTEATOSIS
IN MICE
Swapnil V. Shewale, Elena Boudyguina, Xuewei Zhu , Lulu Shen, Patrick M. Hutchins, Robert
M. Barkley, Robert C. Murphy, John S. Parks.
This chapter is published in J. Lipid Res. 2015 Apr 28. pii: jlr.M059170 (PMID 25921305). Stylistic variations are due to the requirements of the journal.
Swapnil V. Shewale performed the experiments and prepared the manuscript. Dr. John S. Parks acted in an advisory and editorial capacity.
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Botanical oils enriched in n-6 and n-3 fatty acid products of FADS2 are equally effective
in preventing atherosclerosis and hepatosteatosis in mice
Swapnil V. Shewale 1, 3, Elena Boudyguina1, Xuewei Zhu 1, Lulu Shen1,3, Patrick M. Hutchins4,
Robert M. Barkley4, Robert C. Murphy4, John S. Parks1,2.
1Departments of Internal Medicine-Section on Molecular Medicine, 2Biochemistry, and
3Physiology/Pharmacology, Wake Forest School of Medicine, Winston-Salem, NC 27157 and
4Department of Pharmacology, University of Colorado Denver, Aurora, CO 80045
Address correspondence to: Dr. John S. Parks, Department of Internal Medicine-Section on
Molecular Medicine, Wake Forest School of Medicine, Medical Center Blvd, Winston-Salem, NC
27157, USA.
Phone: 336-716-2145; Fax: 336-716-6279; Email: [email protected]
Running title: PUFA-mediated atheroprotection and hepatoprotection in mice
Abbreviations: AD, Atherogenic Diet; AUC, area under the curve; CE, cholesteryl ester;
FADS2, fatty acid desaturase 2 (FADS2) / delta six fatty acid desaturase enzyme; HODES,
Hydroxyoctadecadienoic acids; LDLrKO, LDL receptor KO; LPS, Lipopolysaccharide; Ox-CE,
Oxidized cholesteryl ester-fatty acyl species
3- current address: Lulu Shen, School of Life Science, Jiangsu Normal University. Jiangsu,
China, 221116
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Abstract
Echium oil (EO), which is enriched in 18:4 n-3, the immediate product of FADS2 desaturation of
18:3 n-3, is as atheroprotective as fish oil (FO). The objective of this study was to determine
whether botanical oils enriched in the FADS2 products 18:3 n-6 vs. 18:4 n-3 are equally
atheroprotective. LDL receptor KO mice were fed one of four atherogenic diets containing 0.2%
cholesterol and 10% calories as palm oil (PO) plus 10% calories as: 1) PO, 2) borage oil (BO;
18:3 n-6 enriched), 3) EO; 18:4 n-3 enriched, or 4) FO for 16 weeks. Mice fed BO, EO and FO
vs. PO had significantly lower plasma total and VLDL cholesterol concentrations; hepatic neutral
lipid content and inflammation, aortic CE content, aortic root intimal area and macrophage
content; and peritoneal macrophage inflammation, CE content, and ex vivo chemotaxis.
Atheromas lacked oxidized CEs despite abundant generation of macrophage 12/15
lipooxygenase-derived metabolites. We conclude that botanical oils enriched in 18:3 n-6 and
18:4 n-3 PUFAs beyond the rate-limiting FADS2 enzyme are equally effective in preventing
atherosclerosis and hepatosteatosis compared to saturated/monounsaturated fat due to cellular
enrichment of ≥20 PUFAs, reduced plasma VLDL, and attenuated macrophage inflammation.
Keywords: atherosclerosis, fatty acid/metabolism, inflammation, lipoproteins/metabolism,
macrophages/monocytes, mass spectrometry, VLDL
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Introduction
Despite widespread use of lipid-lowering drugs, cardiovascular disease is still the leading cause
of death in the United States 1. One of the first lines of cardiovascular disease treatment is
reducing total fat intake and replacing saturated fatty acids with polyunsaturated fatty acids
(PUFAs) 2. In particular, n-3 PUFAs from fatty fish or fish oil (FO) reduce the extent of
cardiovascular and other chronic diseases in humans and experimental animals 3-6. The
atheroprotective benefits of fatty fish and FO are attributed to two n-3 PUFAs, eicosapentaenoic
acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3). However, despite the well-
documented health benefits of n-3 PUFAs, consumption is low in the US 7. Furthermore, most
botanical sources of n-3 fatty acids (i.e., flaxseed oil) contain alpha linolenic acid (ALA, 18:3 n-
3), which is inefficiently converted to EPA (4-15%) because of the rate-limiting nature of fatty
acid desaturase 2 (FADS2, i.e., delta-6 desaturase) in the fatty acid elongation and desaturation
pathway 8. Thus, botanical oils enriched in ALA lack the plasma lipid-lowering and
atheroprotective properties observed with FO 9.
Dietary n-6 PUFAs are also atheroprotective in mouse and nonhuman primate models of
atherosclerosis, and in human populations 10-13. Some of the atheroprotection is likely due to
plasma lipid lowering, but there may also be an anti-inflammatory role for n-6 PUFAs through
the generation of prostaglandin E1 (PGE1) from dihomo-gamma-linolenic acid (DGLA, 20:3 n-6),
a potent inhibitor of thromboxane A2 (TXA2) formation that inhibits leukocyte adherence to
endothelial cells 14, 15. However, most sources of botanical n-6 PUFAs are highly enriched in
linoleic acid (LA; 18:2 n-6, ~85-90% of North American dietary PUFA intake) 10, 12. LA also must
be converted by FADS2 to longer-chain bioactive PUFAs, although some bioactive derivatives
of LA are described as both pro- and anti-inflammatory 13.
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One strategy to circumvent the poor conversion of dietary 18-carbon PUFAs to their ≥20 carbon
bioactive products is to identify botanical oils enriched in PUFAs beyond the FADS2 rate-limiting
step of desaturation and elongation. We previously showed that echium oil (EO), which is
relatively enriched in stearidonic acid (SDA, 18:4 n-3), the immediate product of FADS2-
mediated desaturation of ALA, effectively enriches plasma and tissue lipids in EPA 16. In LDL
receptor KO (LDLrKO) mice, isocaloric replacement of palm oil (PO) with EO attenuated
atherosclerosis severity, splenic monocytosis, monocyte influx into aortic intima, and aortic root
intimal macrophage content to an equivalent extent as FO, lending proof of principle for this
strategy 17. However, whether a similar strategy will be atheroprotective for the n-6 PUFA
pathway is unknown.
To address this gap in knowledge, we identified borage oil (BO) as a potential botanical oil to
test this strategy in the n-6 PUFA conversion pathway. BO is enriched (~20%) in gamma
linolenic acid (18:3 n-6; GLA), the immediate product of FADS2-mediated desaturation of LA.
GLA can be elongated to DGLA, a precursor of the anti-inflammatory PGE1, or arachidonic acid
(AA, 20:4 n-6), a precursor of proinflammatory eicosanoid species. The atheroprotective effects
of BO have not been explored, and little is known about its effects on plasma lipids and
lipoproteins. The purpose of this study was to directly compare the atheroprotective potential of
BO with EO and elucidate potential mechanisms for atheroprotection.
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Materials and methods
Dietary oils
The seed oil of Borago officinalis L. (a member of the Boraginaceae family) and the seed oil of
Echium plantagineum L. (a member of the Boraginaceae family) were generous gifts from
Croda Europe Ltd. (Leek, Staffordshire, UK) and authenticated by the Wake Forest University
Center for Botanical Lipids and Inflammatory Disease Prevention. The seed oil of palm, Elaeis
guineensis Jacq (a member of the Arecaceae family) was purchased from Shay and Company
(Portland, OR, USA). For these oils, a certificate of analysis is on file and retention samples are
deposited at the Wake Forest School of Medicine. The fish oil source, Brevoortia tyrannis
Latrobe (a member of the Clupeidae family), was manufactured and generously provided by
Omega Protein (Reidsville, VA, USA) with a report of analysis on file for reference.
Animals and atherogenic diets (AD)
Female LDLrKO (C57BL/6 background) mice (5-6 weeks of age) were purchased from The
Jackson Laboratory (Bar Harbor, Maine, USA). Mice were housed in a specific pathogen-free
facility on a 12h light/dark cycle. All protocols and procedures were approved by the Institutional
Animal Care and Use Committee. Mice were allowed to acclimate for 1-2 weeks during which
time they ate a chow diet. At 7-8 weeks of age mice were randomly assigned to one of four AD
groups (n=15/ diet group) containing 10% calories as PO and 0.2% cholesterol, supplemented
with an additional 10% of calories as 1) PO, 2) BO (18:3 n-6 enriched), 3) EO (18:4 n-3
enriched) or 4) FO (20:5 n-3 and 22:6 n-3 enriched) for 16 weeks. The synthetic ADs were
prepared by the diet kitchen in the Department of Pathology at Wake Forest School of Medicine
as previously described 18. Detailed composition and quality control data for similar ADs have
been published 16.
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Body and organ weights
Mice were weighed every 2-4 weeks. After 16 weeks of AD feeding, body weights were
recorded after a 4-hour fast. Blood was collected via the tail vein at baseline and after 2, 4, 8,
and 16 weeks of AD feeding. Mice were then anesthetized using ketamine-xylazine and
perfused via the left ventricle using cold PBS at the rate of ~3 ml/minute for 3-4 minutes before
organs were harvested. After perfusion, liver wet weights were measured and normalized to
terminal body weight.
Fatty acid analysis
Lipid extraction of lyophilized diets, RBCs, plasma, and liver was performed using the Bligh-
Dyer method 19. The total lipid extracts from plasma and liver were separated into cholesteryl
ester (CE), triglyceride (TG), and phospholipid (PL) fractions by thin-layer chromatography.
Lipids were re-extracted from CE, TG, PL fractions and then trans-methylated using boron
trifluoride 20, and percentage fatty acid composition was quantified by gas-liquid
chromatography (GLC) as described previously 18. To estimate recovery of fatty acids from TLC
and the transmethylation procedure, a known amount of tripentadecanoin (C15:0) and
cholesteryl nonadecanoate (C19:0) were used as internal standards prior to TLC and tri-
heptadecanoin (C17:0) was used as an internal standard prior to transmethylation. All internal
standards were purchased from Nu-Chek-Prep, Inc, (T-145, Ch-818 and T-155). Percentage
loss during TLC and transmethylation was <10% and <15%, respectively. Diet and RBC lipid
extracts were directly trans-methylated for fatty acid analysis.
Plasma lipid and lipoprotein analysis
Plasma was isolated by low-speed centrifugation. Plasma total and free cholesterol (Wako), and
triglyceride (Roche) concentrations were determined using enzymatic assays as described
earlier 21 at baseline and after 2, 4, 8, and 16 weeks of AD feeding. Cholesteryl ester content
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was calculated as (total-free) × 1.67; this calculation corrects for loss of fatty acid during the
cholesterol esterase step of the assay. Data were expressed as area under the curve (AUC),
which integrates plasma lipid concentrations over the 16 weeks, representing a time average
estimate of hypercholesterolemia during the 16 week experiment. Plasma lipoprotein cholesterol
mass distribution was determined using fast protein liquid chromatography (FPLC) fractionation
on a Superose 6 10/300 column (GE Healthcare). Three equal volumes of plasma samples from
each time point (5 mice/group) were pooled and subjected to FPLC fractionation and cholesterol
quantification. VLDL, LDL and HDL cholesterol concentrations were determined and then
expressed as AUC.
In vivo quantification of VLDL TG secretion rate
VLDL TG secretion rate was determined using detergent inhibition of intravascular TG lipolysis
20, 22. Tyloxapol (500 mg/kg body weight) was intravenously injected into anesthetized mice
(n=5/group) fed the AD for 16 weeks and fasted for 4 hours. Plasma TG concentrations before
(0 minutes), and 60, 120 and 180 minutes after injection were determined by enzymatic assay.
TG secretion rate was calculated using linear regression analysis to determine the slope of the
time vs. TG concentration plot for each animal 20.
Liver lipid analysis
Livers were harvested at necropsy, flash frozen in liquid N2, and stored at -80°C. Liver lipids
were quantified using a detergent-based enzymatic assay 23.
Western blot analysis
Nuclear proteins were prepared from frozen livers using ultracentrifugation as described 24, 25.
Equal aliquots (20 μg) of nuclear protein from individual livers were subjected to SDS-PAGE on
4-12% gels and transferred to a PVDF membrane. Immunoblot analyses were performed using
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monoclonal anti-mouse SREBP-1 and rabbit polyclonal SREBP-2 antibodies as described 26.
Anti-SREBP1 and 2 antibodies were generously donated by Dr. Timothy Osborne (Sanford
Burnham Medical Research Institute). Anti-YY1 antibody (Abcam # 43058) was used as a
nuclear loading control. Whole cell lysates were prepared using frozen livers 27 and equal
aliquots of (50 g) of protein from individual livers were subjected to SDS-PAGE on 4-12% gels
and transferred to PVDF membranes. Immunoblot analyses were performed using rabbit
monoclonal anti-FAS antibody (Cell Signalling # 3180) and goat polyclonal anti SCD-1 antibody
(Santa Cruz Biotechnology # 14719).
Atherosclerotic lesion analysis
A subset of mice (n=3/group) was sacrificed after 8 weeks of AD feeding to measure aortic
cholesteryl ester content. In the remaining mice, aortic cholesteryl ester content, aortic root
intimal area, and intimal macrophage content (CD68+) were measured after 16 weeks of AD
feeding 17. Aortic root intimal area was measured with Oil Red O staining.
Aortic oxidized-CE and aortic cholesterol content analysis
At necropsy, aortas were cleaned of visible adventitial fat and placed into a 15 ml etched glass
tissue grinder containing 1 ml of 1:1 MeOH: H2O (v/v) and a known amount of internal standard
5-α cholestane. Aortas were homogenized on ice and neutral lipids were extracted twice using
75:25 isooctane: ethyl acetate (v/v). The combined organic layers were brought to a volume of 2
ml with 75:25 isooctane:ethyl acetate. One ml of the extract was added to an argon-purged
ampule, heat-sealed, and shipped on dry ice to the University of Colorado Denver for LC-
MS/MS analysis. Before analysis, the organic layer was dried under N2. The residue was then
weighed and diluted to 1 g/l with isooctane, and stored at -20°C until analysis of oxidized CE
(ox-CE) using LC/MS (/MS) 28. The remaining 1 ml of the original aortic extract in 75:25
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isooctane: ethyl acetate was used to quantify aortic TC and FC content by GLC 17. Results were
normalized to aortic wet weight.
Peritoneal macrophage studies
Thioglycollate-elicited peritoneal macrophages were isolated and cholesterol content was
measured as described previously 21. Macrophage gene expression was measured after 2
hours of PBS or LPS (200 ng/ml) stimulation 21 and eicosanoids were identified and quantified
using LC/MS/MS in macrophage- conditioned media 29, 30. In vitro macrophage chemotaxis in
response to MCP-1 and MIP-1α was performed in a 48-well microtaxis chamber 31.
Real-time PCR analysis
Total RNA was isolated from mouse macrophages and liver using TRIzol (Invitrogen) and
quantitative real-time RCR was performed to determine gene expression 21. Primer sequences
have been reported previously 20, 27, 32. GAPDH was used as the control for normalization of
results.
Statistics
Data are reported as mean ± SEM. Statistical analyses were performed using one-way ANOVA
and individual paired diet comparisons were made using Tukey's post-hoc analysis. All
statistical analyses were performed using GraphPad Prism software.
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Results
Systemic response to atherogenic diets enriched in n-3 vs. n-6 fatty acid products of
FADS2
Dietary fatty acid compositions are given in Table 1 and show relative enrichment of 18-carbon
fatty acids beyond FADS2 in the BO (19.7% 18:3 n-6) and EO (6% 18:4 n-3) diets. AD feeding
over 16 weeks resulted in uniform food consumption (~3-4 g/day/mouse), body weight gain, and
terminal liver/body weight ratios among all groups (Figure 1). Upon initiation of AD diet feeding,
significant RBC fatty acid enrichment over baseline (chow diet) was evident within 1 week and
appeared to reach equilibrium by 4 weeks (Figure 2). RBC fatty acid compositions reflected
efficient in vivo elongation-desaturation of dietary 18:3 n-6 derived from BO to its longer chain
(20-carbon) counterparts, 20:3 n-6 and 20:4 n-6. Similarly, 18:4 n-3 derived from EO was
elongated-desaturated to 20:5 n-3. These data indicate that dietary enrichment of 18-carbon
fatty acid beyond FADS2 is sufficient to result in membrane enrichment of their respective 20-
carbon chain counterparts. After 4 weeks of diet feeding, compared to chow (baseline), BO-fed
mice had significant RBC fatty acid enrichment in GLA (~5.0% vs. 0.1%), DGLA (~4.0% vs. 1.1
%) and AA (~20.0% vs. 12.0%); whereas EO-fed mice had significant RBC fatty acid
enrichment in ALA (~2.0% vs. 0.4%) and EPA (~3% vs. 2%). FO-fed mice had the greatest
enrichment in EPA (~10% vs. 2%) and DHA (~12% vs. ~10.0 %), whereas PO-fed mice had the
greatest enrichment in OA (~30% vs. 11%) and lowest enrichment in SA (below 10% vs. ~12-
13%).
Fatty acid composition was also measured in plasma and liver CE, TG, and PL fractions (Figure
3). In plasma from BO-fed mice, equivalent (~20%) AA enrichment was observed in CE, TG,
and PL fractions relative to plasma from PO-fed mice (Figure 3 A-C). Plasma neutral lipids from
mice fed BO were also relatively enriched in GLA (CE (~6%) and TG (~15%)), unlike plasma
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PL. In BO-fed livers, AA was modestly, but significantly enriched in PL (~20%), but not in neutral
lipids (below 5%) relative to the other diet groups (Figure 3 D-F). In general, PUFA enrichment
in liver CE and TG was low for all diet groups relative to liver PL.
Plasma lipid and lipoprotein response to dietary BO vs. EO
Chow-fed mice had similar baseline measurements, but after 2 weeks of AD diet feeding,
plasma TC, CE, and TG concentrations increased significantly, peaked by week 4, and
remained elevated over the 16 weeks of diet feeding for PO-fed mice (Figure 4 A-C). This
pattern was significantly attenuated in the other diet groups. BO and EO induced equivalent
cholesterol lowering, whereas FO induced even further TC and CE lowering, compared to PO.
Plasma TG concentrations increased equivalently in PO and BO fed mice after 2 weeks of AD
diet feeding, but stayed at baseline levels in the EO- and FO-fed mice.
A sharp increase in VLDL cholesterol (VLDL-c) concentrations occurred within 2 weeks of AD
feeding that peaked and equilibrated by 4 weeks for PO-fed mice (Figure 5A). VLDL-c
concentrations were significantly and equivalently attenuated in the other diet groups (Figure 5
A, D). LDL-c levels showed a similar pattern; only FO-fed mice had significantly lower LDL-c
concentrations versus PO-fed mice (Figure 5B, E). HDL-c concentrations also increased
sharply within 4 weeks among BO-, EO-, and FO-fed mice, and were relatively stable thereafter.
HDL-c concentrations were significantly lower in PO-fed mice compared to the other groups
(Figure 5C, F); however, HDL-c was <10% of the total pool of plasma cholesterol. Hence, most
of the total cholesterol lowering came from decreases in VLDL-c. BO-induced reduction in
VLDL-c was equivalent to that of EO and FO, but BO did not reduce plasma TG concentrations.
EO, but not BO, diet reduces hepatic VLDL triglyceride secretion rate
We investigated whether the difference in plasma TG concentrations between EO vs. BO-fed
mice could be explained by hepatic TG secretion. Using detergent inhibition of plasma TG
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lipolysis, hepatic TG secretion rates were significantly lower for EO and FO-fed groups
compared to BO and PO-fed mice (Figure 6). These data suggest that the higher plasma TG
concentrations in BO vs. EO fed mice are likely due, in part, to a higher hepatic VLDL TG
secretion rate.
BO- and EO-containing ADs are equally effective in reducing hepatosteatosis
We and others have shown that ADs enriched in FO reduce hepatic lipid content 16, 33-35. In the
present study, ADs containing BO, EO or FO for 16 weeks reduced hepatic total neutral lipid
content (i.e., TG and CE) relative to PO (Figure 7A), but FC and PL contents were similar
among all diet groups. Furthermore, BO and EO were equally effective in reducing hepatic
neutral lipid content. To determine the reason for the decreased hepatic neutral lipid content
with BO and EO feeding, we examined hepatic lipogenic gene expression using quantitative
real-time PCR. Compared to PO, all three ADs significantly lowered SREBP2 mRNA and its
target gene HMG-CoA reductase, but not HMG-CoA synthase (Figure 7B). Although SREBP1-c
mRNA expression levels were similar among all diet groups, expression of its target genes fatty
acid synthase (FAS) and stearoyl-CoA desaturase-1 (SCD1) was significantly reduced in all
three ADs relative to PO (Figure 7B). There were no significant differences among diet groups
in LXRα and ABCA1 expression, whereas PPARα expression was significantly induced in FO-
fed mice (Figure 7B). Since mice consuming the BO diet had reduced hepatosteatosis, similar
to that of mice fed the EO or FO diets, we determined whether BO feeding attenuated hepatic
inflammation as well. mRNA expression of CD68, TNF-α, and MCP-1 were significantly and
equivalently reduced in BO, EO and FO-fed mice compared to those fed PO (Figure 7B).
Expression of other pro-inflammatory cytokines, such as IL-1β, IL-6, and IL-18, and the
alternatively activated macrophage markers arginase-1 and anti-inflammatory cytokine IL-10
were similar among all four diet groups (data not shown). To determine whether FADS2
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products reduce hepatic lipogenic gene expression via attenuation of the SREBP pathway, we
measured accumulation of proteolytically cleaved mature/nuclear SREBP 1 and 2 isoforms in
liver nuclear preparations. Content of nuclear SREBP 1 (but not 2) was significantly reduced in
BO-, EO-, and FO-fed mice relative to PO (Figure 7C). Hepatic protein content of the SREBP-
1c target genes FAS and SCD-1 was also significantly lower in those groups relative to PO
(Figure 7D).
BO and EO are equally atheroprotective
Aortic FC content was comparable among the groups after 8 weeks; however, CE content was
significantly lower (~2 fold lower) in all groups vs. PO-fed mice (Figure 8A). After 16 weeks of
AD feeding, aortic FC and CE content was significantly lower in all groups (~ 2 fold reduction in
FC; ~3-4 fold reduction in CE) vs. PO-fed mice (Figure 8B), indicating that BO, EO and FO
induced equivalent atheroprotection compared to PO. Significant aortic FC accumulation
occurred between 8 and 16 weeks of AD feeding among all groups (~3-4 g/mg at 8 weeks vs.
6-14 g/mg at 16 weeks). CE accumulation increased modestly among all groups (~1-3 g/mg
at 8 weeks vs. ~2-4 g/mg at 16 weeks) relative to the PO group (~4 g/mg at 8 weeks to ~16
g/mg at 16 weeks), indicating a disproportionate increase of CE deposition in PO-fed mice.
Aortic root intimal area was significantly lower (300-400 vs. 700 mm2) in the three PUFA-
containing ADs compared to PO (Figure 8C), as was aortic root intimal macrophage content
(CD68+) (Figure 8D). Collectively, these results show that BO, EO, and FO were equally
effective in reducing aortic atherosclerosis and macrophage content.
Mouse aortas lack of oxidized cholesteryl ester species
Human atheromas contain several distinct families of ox-CE species derived from PUFAs (e.g.
18:2 n-6, 20:4 n-6 and 22:6 n-3) that may play a role in atherogenesis 28. Since three of the four
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ADs showed substantial PUFA enrichment, we examined ox-CE content in mouse aortas after
16 weeks of AD feeding. Aortic cholesteryl ester fatty acyl molecular species reflected the fatty
acid enrichment of the diet; however, aortas had undetectable levels of ox-CE in all diet groups
(Figure 9). The relative abundances of the non-oxidized CE molecular species presented in
Figure 9 insets do not precisely reflect absolute differences in molecular species due to different
electrospray ionization response factors (mass spectrometric parameters) for the
polyunsaturated CEs as previously noted (29). Nonetheless, this raw data reveals dietary
modification of PUFA-containing CE molecular species in aorta tissues reflecting the dietary
fats.
Thioglycollate-elicited peritoneal macrophage eicosanoid release is similar for BO- and
EO-fed mice
We studied the effects of AD feeding on thioglycollate-elicited peritoneal macrophage
eicosanoid biosynthesis. After 16 weeks of AD feeding, thioglycollate-elicited peritoneal
macrophages were incubated ± LPS for 2 hours before media eicosanoids were quantified. This
model of stimulated eicosanoid biosynthesis has been recently reported, including details of
gene expressions related to PUFA metabolism stimulated by LPS (30). Among the eicosanoids
measured, 12/15 lipoxygenase-derived LA metabolites 9 and 13 HODE were most abundant
(Figure 10). Furthermore, the type of dietary fat had little impact on production of thioglycollate-
elicited peritoneal macrophage eicosanoid species in the basal state or after LPS stimulation.
Only in the FO group were significant reductions observed for generation of TXB2, PGE2, and 12
hydroxyeicosatetraenoic acids after LPS stimulation (~2-4 fold lowering vs. PO, EO and BO).
These data indicate that LPS stimulation revealed little differences in thioglycollate-elicited
peritoneal macrophage eicosanoid biosynthesis among BO-, EO-, and PO-fed mice.
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BO and EO attenuate macrophage inflammatory response to LPS
HODEs are natural ligands for macrophage PPARγ, which, when activated, can result in down-
regulation of the canonical NF-κB pathway 36, 37. Thus, we measured expression of NF-κB target
genes after LPS-induced activation in thioglycollate-elicited PMs isolated from mice after 16
weeks of AD feeding. Basal (-LPS) gene expression of the pro-inflammatory cytokines IL-6, IL-
1β, TNF-α, and chemokine MCP-1 was comparable among all groups (Figure 11A). LPS-
treated macrophages from mice fed BO, EO, and FO vs. PO had decreased mRNA expression
of IL-6, IL-1β and MCP-1, whereas TNF-α expression was significantly higher (Figure 11A).
However, 6 hours after LPS stimulation, TNF-α mRNA expression was significantly lower in
macrophages from BO, EO and FO vs. PO-fed mice (data not shown), similar to the results for
IL-6, IL-1β and MCP-1 at 2 hours. mRNA abundance for the alternatively activated macrophage
markers arginase 1 and CD206 was not different among groups (data not shown). Collectively,
these data indicate that BO attenuates macrophage LPS-induced pro-inflammatory gene
expression to the same extent as EO and FO, without affecting the gene expression of
alternative-activated macrophage markers.
BO and EO equally attenuate macrophage CE accumulation and chemotaxis
Given the equivalent effectiveness of BO in attenuating aortic CE and macrophage content, we
sought to determine whether macrophages from BO-fed mice contributed to reduced aortic
atherosclerosis via reduced macrophage foam cell formation. We used thioglycollate-elicited
PMs (as a surrogate for aortic macrophages) from mice after 8 and 16 weeks of AD exposure to
estimate cholesteryl ester content. At 8 weeks, macrophage CE content ranged from 5 to 20
g/mg among the groups, but only the FO-fed mice had significantly lower macrophage CE
content compared to PO-fed mice (Figure 11B). After 16 weeks of AD feeding, macrophage CE
content increased considerably (~2-8 fold) compared to 8 weeks and was significantly
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attenuated in BO, EO and FO vs. PO-fed mice (Figure 11B). We next investigated whether
reduced macrophage chemotaxis might partially explain the reduction in aortic root macrophage
content in BO-, EO-, and FO-fed mice. All 3 PUFA containing ADs were equally effective in
reducing chemotaxis to MIP-1α and MCP-1 compared to PO-fed mice (Figure 11C).
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Discussion
In the current study, we tested the hypothesis that botanical oils enriched in 18:3 n-6 (BO) and
18:4 n-3 (EO) PUFAs beyond the rate-limiting FADS2 enzyme are equally atheroprotective
compared to saturated/monounsaturated fat (PO) in LDLrKO mice. Although BO differs from EO
and FO in its inability to lower plasma triglycerides, BO and EO were comparable in their ability
to lower plasma cholesterol concentrations, especially VLDL-c. Additionally, BO was as effective
as EO and FO in alleviating hepatic steatosis, and in regulating hepatic lipogenic and
inflammatory gene expression compared to PO. As a result, BO and EO resulted in significant
atheroprotection relative to PO at early (8 weeks) and advanced (16 weeks) atherosclerotic
stages. We also report that ox-CEs were undetectable in early and advanced atherosclerotic
arteries, suggesting that ox-CEs play a minimal role in murine atherosclerosis progression.
Additionally, BO and EO significantly and equivalently attenuated macrophage inflammatory
gene expression, CE content, and chemotaxis. BO had these atheroprotective outcomes
despite significant enrichment of RBC membranes, and plasma and liver lipids with 20:4 n-6, a
precursor for several proinflammatory eicosanoids. Our results support the conclusion that
botanical oils enriched in 18:3 n-6 and 18:4 n-3 PUFAs beyond the rate-limiting FADS2 enzyme
are equally atheroprotective and hepatoprotective compared to saturated/monounsaturated fat.
Although previous studies in nonhuman primates and mice have demonstrated n-6 PUFA-
mediated atheroprotection, these studies used dietary fats primarily enriched in LA 38-40. Our
study focused on the hypothesis that dietary enrichment with n-6 PUFAs beyond FADS2 (i.e.,
GLA) would be as atheroprotective as EO, which we have previously demonstrated equal to FO
in preventing atherosclerosis progression 17, 41. On the other hand, flaxseed oil, which is
enriched in ALA, a substrate for FADS2, was not as atheroprotective as FO; this outcome
occurred despite significant enrichment of liver phospholipids with EPA and was likely due to the
lower plasma LDL-c concentrations in the FO vs. flaxseed oil group 9. The combined results of
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the flaxseed oil and EO studies support our hypothesis that dietary enrichment in n-3 PUFAs
beyond FADS2 is atheroprotective.
Our current results show that this is also true for the n-6 pathway of fatty acid elongation-
desaturation. BO, which is enriched in GLA, was as atheroprotective as EO and FO despite its
significant enrichment of RBCs, and plasma and liver lipid fractions with AA, a fatty acid
precursor to pro-inflammatory leukotrienes and prostaglandins 42. Concerns that elevated
membrane AA may result in increased cellular inflammation that exacerbates atherosclerosis
lack support in human studies 42. Moreover, LA-enriched diets have not enriched AA in plasma
and tissue lipid fractions 40, 43, 44, likely due to inefficient FADS2 conversion of LA to AA 45, 46. In
addition, a recent meta-analysis of 13 cohort studies (involving 310,602 individuals and 12,479
coronary heart disease events) revealed an inverse association between dietary LA intake and
coronary heart disease risk, such that, a 5% increase in energy intake from LA was associated
with a 10% and 13% lower risk of coronary heart disease events and deaths, respectively 12.
Collectively, these results suggest that increased consumption of dietary LA and GLA is not
harmful and is potentially atheroprotective in the general population. Our results also suggest
that assessing cardiovascular risk and inflammatory potential by dietary n-3/n-6 PUFA ratio may
be an oversimplification that does not take into account differences between 18 vs. ≥20 carbon
PUFAs.
A recent study reported a single nucleotide polymorphism (rs174537) in the FADS1/2 gene
cluster that affects plasma AA levels 47. The GG allele, which is nearly twice as frequent in
African Americans as in European Americans, was associated with a small (~3%) but
statistically significant enrichment in plasma AA. A subsequent study demonstrated that GG
homozygotes for the rs174537 allele had increased plasma AA enrichment and production of
LTB4 and 5-hydroxyeicosatetraenoic acids in zymosan- stimulated blood, suggesting that
genetic polymorphisms may influence tissue AA content and inflammatory response to external
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pathogens 48. Thus, individuals harboring rs174537 homozygous GG alleles may be
hyperresponsive to diets enriched in LA and GLA. Further studies are required to determine
whether this potential hyperresponsiveness affects coronary heart disease risk.
Our results also suggest that BO and EO slow atherosclerosis progression in multiple ways –
including reduced plasma VLDL-c concentrations, macrophage cholesterol content,
inflammatory gene expression, and decreased macrophage migration towards a chemokine
gradient. Plasma VLDL-c reduction likely had the greatest influence on atherosclerosis outcome
in this study, since VLDL-c concentrations are the best lipoprotein/lipid predictor of aortic root
atherosclerosis in LDLrKO mice 49 and we observed a strong positive association between
plasma VLDL-c and aortic CE content (r2=0.88; p<0.0001, data not shown). LDL-c
concentrations were similar among PO-, BO-, and EO-fed mice, suggesting they had a minimal
effect on atheroprotection in BO and EO-fed mice. Plasma HDL-c was significantly elevated in
all three diet groups and may have contributed to atheroprotection relative to the PO group;
however, only a small fraction (~10%) of plasma cholesterol was distributed in HDL particles,
making this a less likely possibility. In other studies investigating the influence of dietary n-3 and
n-6 PUFA on atherogenesis, LDL was the predominant plasma atherogenic lipoprotein 9, 38.
Differences in plasma apoB lipoprotein response (VLDL vs. LDL) among these studies are likely
due to genetic background (LDLrKO vs. apoB100 only-LDLrKO) and diet composition (0.02 vs.
0.2% cholesterol; 10% vs. 20% calories as fat).
A significant driver of atherogenesis in nonhuman primates and LDLrKO mice is hepatic
production of saturated and monounsaturated CEs that are core constituents of secreted VLDL
particles 11. Plasma VLDL particles undergo intravascular metabolism to become LDL particles,
a major atherogenic lipoprotein particle in plasma. Deletion of the cholesterol esterification
enzyme steroyl O-acyltransferase 2 (SOAT2) strikingly reduces atherosclerosis regardless of
dietary fat saturation 38, supporting a critical role for SOAT2 in the generation of atherogenic
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CEs in apoB-containing lipoproteins, such as VLDL and LDL. Hepatic CE content was
significantly and equivalently reduced in mice fed BO and EO, suggesting the production of
atherogenic CEs was blunted in these mice compared to those fed PO. This likely contributed to
lower VLDL-c concentrations for the EO and BO groups, and reduced monounsaturated 18:1 n-
9 CE species in plasma at the expense of FADS2-derived CEs.
One of the earliest events in atherogenesis is arterial retention of apoB lipoproteins by
proteoglycans 50. A recent study showed that SOAT2 KO vs. WT mice or mice fed FO vs.
monounsaturated fat had plasma LDL enriched in PUFA CE species and bound with less affinity
to proteoglycans, suggesting a mechanism by which apoB lipoproteins from mice fed dietary
PUFA may be less atherogenic 51. Although PUFA lipid species are more easily oxidized than
their saturated and monounsaturated counterparts 52, 53 and ox-CE species have been identified
in human tissue samples from endarterectomies 28, no arterial ox-CEs were detected in our
study, despite significant enrichment of circulating and tissue lipids with AA in the BO group
(Figures 2, 3, and 9). Thus, ox-CEs did not appear contribute significantly to atherogenesis in
our study.
CE-loaded macrophages are a hallmark of atherosclerosis, which result from chemokine-
induced chemotaxis of monocytes to atherosclerotic lesions, differentiation of monocytes into
macrophages expressing scavenger receptors, and unregulated uptake of modified apoB
lipoproteins and apoB lipoprotein-proteoglycan complexes 54. In addition to reduced aortic
cholesterol and aortic root intimal area in mice fed BO and EO vs. PO, we also observed
decrease aortic root macrophage (CD68+ cells) content and decreased macrophage
chemotaxis in vitro. Thioglycollate-elicited peritoneal macrophages isolated from BO and EO fed
mice also had reduced sterol content and inflammatory response to LPS compared to their PO-
fed counterparts. Macrophage FC accumulation resulting from genetic deletion of macrophage
efflux genes ABCA1 and ABCG1 results in hyper-responsiveness to proinflammatory stimuli and
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increased chemotaxis in vitro and in vivo 21, 55. However, in this study, the cholesterol elevation
was due to CE, not FC, and there was no increase in macrophage ABCA1 and ABCG1 gene
expression among diet groups (data not shown). These results suggest that decreased uptake
of cholesterol from apoB lipoproteins likely explained the decreased macrophage atherogenic
phenotype for mice fed EO and BO.
Diets enriched in n-3 PUFAs reduce hepatosteatosis 9, 16, 33, 56, unlike those containing n-6
PUFAs 27, 56. The reduced hepatic neutral lipid content in animals fed n-3 PUFAs is primarily
mediated through decreased hepatic lipogenesis and is usually accompanied by reduced
hepatic TG secretion 34, 57. In vitro, n-3 and n-6 PUFAs suppress SREBP-1c gene transcription
58, 59, decreasing hepatic lipogenesis, by competing with activators of LXR, a potent inducer of
hepatic TG synthesis 58, 60. n-3 and n-6 PUFAs also increase mRNA degradation 61, inhibit the
proteolytic processing of SREBP-1c in vitro 59, and accelerate the degradation of nuclear
SREBP-1c 62, reducing lipogenesis. Thus, we hypothesize that BO, but not the other n-6 PUFA-
enriched diets (27,56), reduces hepatosteatosis relative to diets containing
saturated/monounsaturated fatty acids via its ability to enrich liver lipids in AA through
elongation and desaturation of GLA. Many n-6 PUFA-enriched botanical oils contain LA as the
predominant fatty acyl species, accounting for 85-90% of n-6 PUFA consumption in the US 10.
However, as discussed above, diets enriched in LA do not result in plasma and tissue AA
enrichment 44, 63. Genetic deletion of Elovl5, the gene encoding the enzyme that elongates 18:3
n-6 to 20:3 n-6 and 18:4 n-3 to 20:4 n-3, results in diminished hepatic lipid AA and DHA and
increased neutral lipid storage 64. Feeding Elovl5 knockout mice AA or DHA rescued the
hepatosteatosis phenotype by decreasing nuclear SREBP-1c and de novo lipogenesis with no
changes in mRNA or membrane-bound SREBP-1c, supporting a role for AA in blocking SREBP-
1c cleavage and activation. In our study, liver membrane (i.e., phospholipid) AA content was
elevated, nuclear SREBP-1 content was reduced, and SREBP-1c targeted genes (FAS, SCD-1)
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were reduced in BO vs. PO fed mice, supporting a role for elevated AA in reducing
hepatosteatosis in BO-fed mice. In another study, BO reduced ethanol-induced hepatosteatosis
65, suggesting that BO protects against hepatosteatosis regardless of the method of induction.
How elevated liver AA inhibits the proteolytic processing of membrane SREBP-1c is unclear, but
may be related to fluidity of the endoplasmic reticulum membrane. Regardless of the detailed
mechanism for decreasing hepatic lipogenesis, our study reveals a distinct advantage of BO in
reducing hepatosteatosis and atherosclerosis in contrast to other n-6 PUFA-enriched botanical
oils that are atheroprotective, but do not prevent hepatosteatosis.
Although BO, EO, and FO all reduced hepatosteatosis comparably relative to PO, BO did not
reduce hepatic TG secretion nor plasma TG concentrations, whereas EO and FO did (Figure 6).
FO consistently reduces hepatic TG secretion, likely due to reduced hepatic lipogenesis 34, 41.
This paradox is likely due to the fact that only a small fraction of hepatic TG is mobilized for
secretion; therefore, a large decrease in hepatic TG content does not necessarily result in
decreased TG secretion. For example, knockdown of SCD-1 with a targeting anti-sense
oligonucleotide resulted in a 90% reduction in hepatic TG content, but did not affect hepatic TG
secretion compared to mice treated with a non-targeting ASO 66. We also observed a 50%
decrease in newly synthesized TG secreted from livers of monkeys fed FO vs. lard diets,
although liver TG synthesis was similar between diet groups 34. These combined results suggest
a unique secretory pool of TG that may be regulated differently than the bulk TG storage pool in
hepatocytes.
Collectively, our results support the hypothesis that dietary enrichment with FADS2 fatty acid
products, such as SDA and GLA, results in membrane and plasma lipid enrichment in EPA and
AA, which in turn, is associated with reduced plasma lipids, atherosclerosis, and
hepatosteatosis. This hypothesis was based on data in humans and animal models showing
that conversion of 18-carbon PUFAs to ≥20 carbon PUFAs is inefficient, and that bypassing the
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rate-limiting FADS2 step is possible by feeding botanical oils enriched in FADS2 products.
Furthermore, allowing for body surface area differences between mice and humans 67,
achieving this dose of botanical oils in the diet is feasible. For example, our ADs contained BO
and EO as 10% energy, which corresponds to a human equivalent dose of 0.81% energy (10%
× 3/37; 67), well within the range of n-3 PUFA doses administered in many randomized clinical
trials 68 and the American Heart Association’s recommended n-3 PUFA intake for individuals
with documented coronary heart disease 69. While replacing dietary saturated fat with PUFA is
viewed as atheroprotective in general, our study suggests a more targeted approach of dietary
PUFA replacement using known biochemical pathways may enhance the beneficial outcomes of
increased dietary PUFA consumption.
Acknowledgments
Anti-SREBP1 and 2 antibodies were generously donated by Dr. Timothy Osborne (Sanford
Burnham Medical Research Institute). We gratefully acknowledge Karen Klein (Biomedical
Research Services and Administration, Wake Forest School of Medicine) for editing the
manuscript. This work was supported by grants from National Institute of Health P50 AT002782
and R01HL119962 (to JSP).
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Figures
Fig. 1. Body weight gain and terminal liver/body weight ratios. (A) Body weight gain was
monitored periodically from baseline (chow diet) to 16 weeks of feeding the indicated
atherogenic diets. (B) Mice were then euthanized and liver wet weights were measured and
normalized to terminal body weight. Data are expressed as mean ± SEM; n=15/diet group. No
significant differences were found by one-way ANOVA.
Fig. 2. RBC fatty acid (FA) composition. RBCs were harvested from mice consuming the
indicated atherogenic diets from baseline (chow diet) to 10 weeks; percentage of fatty acid
distribution was measured as described in the methods. Data for individual fatty acids are
expressed as percentage composition of total fatty acids. An equal volume pool of RBCs from 8
mice/diet group/week was used for analysis. Horizontal line denotes extrapolation of chow data
(0 weeks) across the 10-week period.
Fig. 3. Percentage fatty acid composition of plasma and liver lipids. LDLrKO mice were fed
the indicated atherogenic diets for 16 weeks before harvesting plasma and liver for fatty acid
analysis. Data for individual fatty acids are expressed as percentage (mean ± SEM) composition
of total fatty acids; n=3/diet group. Bars with different letters denote significant (p<0.05)
differences among diet groups by one-way ANOVA and Tukey’s post-hoc analysis.
Fig. 4. Plasma lipid concentrations. Fasting (4h) plasma (A) total cholesterol (TC), (B)
cholesteryl ester (CE), and (C) triglyceride (TG) concentrations in LDLrKO mice during a 16
week AD feeding were measured by enzymatic assays (n=15). CE= (TC-FC)*1.67. Plasma TC,
CE and TG concentrations were integrated over the 16-week study and expressed as area
under the curve (AUC) (D, E, F). Data are expressed as mean ± SEM, n=15/diet. Groups with
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different letters are significantly different (p<0.05) by one-way ANOVA and Tukey’s post-hoc
analysis.
Fig. 5. Plasma lipoprotein cholesterol distribution. LDLrKO mice were fed the indicated
atherogenic diets for 16 weeks. Fasted (4h) plasma was harvested and fractionated by FPLC
and cholesterol distributions among VLDL (A), LDL (B) and HDL (C) fractions was measured.
Plasma VLDL-c (D), LDL-c (E) and HDL-c (F) concentrations were integrated over the 16-week
study and expressed as area under the curve (AUC). Data are expressed as mean ± SEM, n=3
equal volume pooled samples from 5 mice/group. Groups with different letters are significantly
different (p<0.05) by one-way ANOVA and Tukey’s post-hoc analysis.
Fig. 6. Hepatic VLDL-TG secretion rate. (A) Plasma TG levels were measured by enzymatic
assay before (0 min) and after (60, 120, 180 min) intravenous Triton® injection. (B) Hepatic TG
secretion rate during the 3h experiment was calculated for each animal as the slope of the
regression line. Data are plotted as mean ± SEM, n=4-5. Groups with different letters are
significantly different (p<0.05) by one-way ANOVA and Tukey’s post-hoc analysis.
Fig. 7. Hepatic response to atherogenic diets. LDLrKO mice were fed the indicated
atherogenic diets for 16 weeks before liver was harvested to measure lipid content and gene
expression. (A) Hepatic lipid content. Neutral lipid= CE+TG. (B) Hepatic mRNA expression of
genes involved in cholesterol biosynthesis, lipogenesis, and inflammation. (C) Nuclear
accumulation of mature SREBP-1c and 2 isoforms in liver. (D) Hepatic FAS and SCD-1 protein
content. Data are expressed as mean ± SEM, n=5. Groups with different letters are significantly
different (p<0.05) by one-way ANOVA and Tukey’s post-hoc analysis.
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Fig. 8. Aortic atherosclerosis. LDLrKO mice were fed the indicated atherogenic diets for 16
weeks before aortas and hearts were harvested for atherosclerosis quantification. Aortas were
lipid extracted for quantification of total cholesterol and free cholesterol (FC) content using gas-
liquid chromatography. Cholesterol ester (CE) content was calculated as (TC-FC) x1.67. (A)
Aortic FC and CE content of mice fed PO, BO, EO and FO for 8 wks or (B) 16 wks. Each data
point represents an individual mouse aorta. (C) Quantification of aortic root Oil Red O positive
intimal area (lesion area) and representative Oil Red O-stained aortic root sections. (D)
Quantification of percentage aortic root lesional area occupied by CD68+ cells and
representative CD68+ immunohistochemically stained aortic root sections. Each point
represents the average lesion area of 6-8 sections per mouse. Horizontal lines denote the mean
for each diet group. Groups with different letters are significantly different (p<0.05) by one-way
ANOVA and Tukey’s post-hoc analysis.
Fig. 9. Mouse atherosclerotic plaque oxidized cholesteryl ester analysis. LDLrKO mice
were fed the indicated atherogenic diets for 16 weeks before aortas were harvested for oxidized
CE analysis using normal phase-HPLC-MS/MS. Ion chromatograms of cholesteryl ester (2-4
min elution) and oxidized cholesteryl esters (6-36 min elution) for one aorta from each diet
group. Inset presents the raw mass spectrometric data corresponding to CE molecular species
labeled with m/z values and their acyl components, denoted by total acyl carbons and double
bonds in this qualitative study. No oxCE species were detectable for any of the four diet groups.
Fig. 10. LPS-stimulated eicosanoid release from peritoneal macrophages. LDLrKO mice
were fed the indicated atherogenic diets for 16 weeks. Mice were then injected with
thioglycollate in the peritoneal cavity and macrophages were isolated 4 days later as described
in the methods. The macrophages were cultured for 3 hours in serum-free medium and then
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treated with LPS (200 ng/ml) or PBS (-LPS) for 2 hours before 1 ml media were collected for
eicosanoid quantification by LC-MS/MS analysis. Eicosanoids below the minimum detection
level are not shown. Data are expressed as mean ± SEM, n=5/group. Groups with different
letters are significantly different (p<0.05) by one-way ANOVA and Tukey’s post-hoc analysis.
Fig. 11. Macrophage inflammation, foam cell formation, and chemotaxis. LDLrKO mice
were fed the indicated atherogenic diets for 8 or 16 weeks before thioglycollate-elicited
macrophages were isolated. (A) Inflammatory gene expression was measured using RT-PCR
after 2 hour treatment with LPS (200 ng/ml) or PBS (-LPS) in thioglycollate-elicited peritoneal
macrophages. (B) Peritoneal macrophage cholesteryl ester content was measured using gas-
liquid chromatography after 8 and 16 weeks of atherogenic diet feeding. (C) Ex vivo chemotaxis
of thioglycollate-elicited peritoneal macrophages towards MCP-1 and MIP-1α was measured
after 16 weeks of atherogenic diet feeding. Data are expressed as mean ± SEM, n=5. In panels
B and C, data points for individual mice are also shown. Groups with different letters are
significantly different (p<0.05) by one-way ANOVA and Tukey’s post-hoc analysis.
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TABLE 1. Fatty acid composition (% FA) and total energy equivalence (% EE) of
individual fatty acids in each atherogenic diet
Palm oil (PO) Borage oil (BO) Echium oil (EO) Fish oil (FO)
Fatty Acid % FA % EE % FA % EE % FA % EE % FA % EE
Palmitic Acid
(C16:0)
43.2 8.64 24.5 4.9 25.6 5.12 30.6 6.12
Palmitoleic Acid
(C16:1)
0.37 0.074 0.3 0.06 0.4 0.08 4.2 0.84
Stearic Acid
(C18:0)
4.5 0.90 4.4 0.88 4.1 0.82 4.5 0.9
Oleic Acid
(C18:1 n-9)
37.3 7.46 24.1 4.82 26.1 5.22 24.9 4.98
Linoleic Acid
(C18:2 n-6), LA
11.1 2.22 17.4 3.48 15.4 3.08 8.2 1.64
α-Linolenic Acid
(C18:3 n-3), ALA
0.37 0.074 0.5 0.11 13.8 2.76 1 0.2
γ-Linolenic Acid
(C18:3 n-6), GLA
19.7 3.94 5.0 1.0
Stearidonic Acid
(C18:4 n-3), SDA
0.2 0.04 6.0 1.2 1.5 0.3
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Euric acid
(C22:1 n-9)
2.6 0.52 0.2 0.04
Eicosapentaenoic
Acid (C20:5 n-3),
EPA
0.1 0.02 0.3 0.06 0.3 0.06 6.9 1.38
Docasahexaenoic
Acid (C22:6 n-3),
DHA
0.2 0.04 0.3 0.06 0.3 0.06 7 1.40
Diets contained 0.2% cholesterol + 10% calories as Palm Oil (PO) + 10% calories as: PO,
Borage oil (BO), Echium oil (EO) or Fish oil (FO). Percent (%) fatty acid composition of PO, BO,
EO and FO diets determined using gas-liquid chromatography. % EE for individual fatty acid
was calculated using total energy derived from fatty acids (i.e. 20%) / diet and % fatty acid
composition of respective diet.
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CHAPTER III
IN VIVO ACTIVATION OF LEUKOCYTE GPR120 BY POLYUNSATURATED FATTY ACIDS
HAS MINIMAL IMPACT ON ATHEROSCLEROSIS IN LDLrKO MICE
Swapnil V. Shewale, Amanda L. Brown, Xin Bi, Elena Boudyguina, Martha Alexander-Miller, Da
Young Oh, Jerrold M. Olefsky, John S. Parks.
This manuscript will be submitted to Circulation Research.
Stylistic variations are due to the requirements of the journal.
Swapnil V. Shewale performed the experiments and prepared the manuscript.
Dr. John S. Parks acted in an advisory and editorial capacity.
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In vivo activation of leukocyte GPR120 by polyunsaturated fatty acids has minimal
impact on atherosclerosis in LDLrKO mice
Shewale: Leukocyte GPR120 and PUFA-induced atheroprotection
Swapnil V. Shewale 1, 2, Amanda L. Brown1, Xin Bi1, Elena Boudyguina1, Martha Alexander-
Miller3, Da Young Oh4, Jerrold M. Olefsky4, John S. Parks1.
1Departments of Internal Medicine/Section on Molecular Medicine, 2Physiology/Pharmacology,
and 3Microbiology and Immunology, Wake Forest School of Medicine, Winston-Salem, NC
27157 and 4Department of Medicine, University of San Diego, La Jolla, CA 92093
Address correspondence to:
Dr. John S. Parks,
Department of Internal Medicine/Section on Molecular Medicine,
Wake Forest School of Medicine,
Medical Center Blvd, Winston-Salem, NC 27157, USA.
Phone: 336-716-2145;
Fax: 336-716-6279;
Email: [email protected]
Total word count: XXXX/7000 (including Title Page, Abstract, Text, References, Tables and
Figures Legends)
Subject Codes:
[90] Lipid and lipoprotein metabolism
[130] Animal models of human disease
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ABSTRACT
Rationale: G protein-coupled receptor 120 (GPR120) activation by n-3 polyunsaturated fatty
acids (PUFAs) attenuates NF-κB inflammatory signaling. However, the impact of GPR120
expression on atherosclerosis is unknown.
Objective: To determine whether in vivo activation of leukocyte GPR120 by n-3 vs. n-6 PUFA is
atheroprotective.
Methods and Results: Leukocyte GPR120 wildtype (WT) or knockout (KO) mice in the LDL
receptor knockout background were generated by bone marrow transplantion. Mice were fed
one of the four atherogenic diets containing 0.2% cholesterol and 10% calories as palm oil (PO)
+ 10% calories as: 1) PO, 2) fish oil (FO; 20:5 n-3 and 22:6 n-3 enriched), 3) echium oil (EO;
18:4 n-3 enriched), or 4) borage oil (BO; 18:3 n-6 enriched) for 16 weeks. Plasma lipids and
lipoproteins, hepatic lipid content, neutrophilia, monocytosis, aortic root monocyte recruitment,
and aortic atherosclerosis were analyzed. Compared to PO, mice fed BO, EO and FO had
significantly reduced plasma cholesterol, triglycerides, VLDL cholesterol, hepatic neutral lipid,
and atherosclerosis that were equivalent for WT and KO mice, demonstrating that leukocyte
GPR120 expression did not affect these outcomes. In BO, EO and FO, but not PO-fed mice,
lack of leukocyte GPR120 resulted in neutrophilia, pro-inflammatory Ly6Chi monocytosis,
increased monocyte recruitment into aortic roots, and increased hepatic inflammatory gene
expression.
Conclusions: We conclude that leukocyte GRP120 expression has minimal effects on dietary
PUFA-induced plasma lipid/lipoprotein reduction and atheroprotection, and that there is no
distinction between n-3 vs. n-6 PUFAs in activating anti-inflammatory effects of leukocyte
GPR120 in vivo.
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Keywords:
GPR120, atherosclerosis, n-3 PUFA, n-6 PUFA, hepatosteatosis, monocytes
Non-standard Abbreviations and Acronyms:
AD: Atherogenic diet
AP-1: activator protein-1
BMT: Bone marrow transplantation
CMPs: common myeloid progenitors
CVD: cardiovascular disease
CE: Cholesteryl ester
ECM: Extracellular matrix
FADS2: fatty acid desaturase 2 (FADS2) / delta six fatty acid desaturase (D6D) enzyme
FFAR: free fatty acid receptor
GPR120: G protein coupled receptor 120
HSCPs: hematopoeitic stem cell precursor (HSCPs)
IKK: I kappaB kinase
JNK: c-Jun N-terminal kinase
PUFA: polyunsaturated fatty acids
PGE1/2: prostaglandin E1/2
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LDLrKO: LDL receptor knockout mice
LP: Lipoproteins
LPS: Lipopolysaccharide
MMP’s: matrix metalloproteases
n-3/n-6 PUFA: omega-3/omega-6 polyunsaturated fatty acids
NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells
SOAT2: cholesterol esterification enzyme steroyl O-acyltransferase 2
SREBP: Sterol responsive element binding protein
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INTRODUCTION
Deorphanization of free fatty acid receptors (FFARs) has allowed further understanding
of free fatty acids (FA) as signaling molecules. These class A, G-protein coupled receptors
(GPRs) include GPR40 (FFAR1), GPR43 (FFAR2), GPR41 (FFAR3), GPR84 and GPR120
(FFAR4) that are activated by short, medium or long chain FA1-6. GPR120 is highly expressed in
intestine, adrenals, lung, adipose tissue, and macrophages, and is described as the n-3
polyunsaturated fatty acid (PUFA) receptor. Upon activation by n-3 PUFA, GPR120 inhibits
transforming growth factor beta-activated kinase 1 (TAK1) activation, resulting in attenuation of
IKKB/NF-κB and JNK/AP1 signaling7. GPR120 expression regulates obesity in mice and
humans; a non-synonymous mutation (p.R270H) inhibited GPR120 signaling activity, resulting
in increased risk of obesity in European populations8. In vivo, selective activation of GPR120 by
n-3 PUFAs is anti-inflammatory and insulin sensitizing7, 8. High fat diet-fed GPR120 knockout
(KO) mice vs. WT counterparts supplemented with n-3 PUFA or a selective GPR120 agonist
(cpdA)9 have: 1) increased adipose tissue F4/80+ macrophage infiltration and pro-
inflammatory/M1 gene expression, 2) increased M1 gene expression in LPS-stimulated
peritoneal macrophages, and 3) increased insulin resistance. Collectively, these findings
highlight the anti-inflamatory potential of macrophage GPR120 activation by n-3 PUFA.
However, the impact of GPR120 expression on atherosclerosis progression, particularly in the
context of dietary fatty acid composition, is unknown.
One of the first lines of cardiovascular disease (CVD) treatment is reducing total fat
intake and replacing saturated fatty acids with PUFAs10. n-3 as well as n-6 PUFAs are
atheroprotective in mice, non-human primates, and humans11-20. In humans, dietary n-3 PUFAs,
eicosapentaenoic acid (EPA; 20:5 n-3) and docasahexaenoic acid (DHA; 22:6 n-3) found in fish
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oil (FO), are anti-inflammatory and lower plasma triglycerides (TG), but not plasma LDL
cholesterol, a primary risk factor for atherosclerosis in humans 21, 22. However, FO consumption
is low in the USA 15. Dietary linoleic acid (LA; 18:2 n-6) is cardioprotective in humans, such that,
a 5% increase in energy intake from LA is associated with a 10% and 13% lower risk of
coronary heart disease events and deaths, respectively 18. LA-enriched diets in nonhuman
primates and mice also are atheroprotective compared to saturated/monounsaturated diets 23-25.
Concerns that increased n-6 PUFA consumption may result in elevated membrane arachidonic
acid (AA; 20:4 n-6), increased cellular inflammation, and atherosclerosis exacerbation lack
support in humans 26. Moreover, LA-enriched diets do not enrich AA in plasma and tissue lipid
fractions 25, 27, 28, likely due to inefficient fatty acid desaturase-2 (FADS2) conversion of LA to AA
29, 30. Although some of the atheroprotection by n-6 PUFAs is likely due to plasma lipid lowering,
diets enriched in desaturation (via FADS2)-elongation products of LA, such as dihomo gamma
linolenic acid (DGLA; 20:3 n-6), have anti-inflammatory potential through generation of
prostaglandin E1 (PGE1), a potent thromboxane A2 inhibitor that reduces leukocyte endothelial
cells adherence31, 32. Currently, in North American diets, plant-derived LA and alpha linolenic
acid (ALA; 18:3 n-3) constitute the majority of PUFA intake 16, 18. Despite high consumption of
LA and ALA, tissue enrichment of their longer chain bioactive products, AA and EPA,
respectively, is limited due to inefficiency of the rate limiting FADS2 enzyme (16,17). To
circumvent this, we have identified botanical oils enriched in n-3 or n-6 PUFAs beyond FADS2,
18:4 n-3 (stearidonic acid; SDA)-enriched echium oil (EO) and 18:3 n-6 (gamma linolenic acid;
GLA)-enriched borage oil (BO). We previously demonstrated that EO and BO are as
atheroprotective and hepatoprotective as FO, compared to a diet enriched in
saturated/monounsaturated fat (PO)33.
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Based on available information, we hypothesized that leukocyte GPR120 (L-GPR120)
activation contributes to the atheroprotective effects of n-3, but not n-6, PUFAs. Selective
activation of L-GPR120 by n-3 PUFAs should result in decreased leukocyte inflammation and
atherosclerosis. We tested this hypothesis by feeding PO, FO, EO or BO diets to leukocyte
GPR120 WT and KO mice in the C57BL/6-LDLrKO background generated by bone marrow
transplantation (BMT). To our knowledge, this is the first study to determine the in vivo role of
leukocyte GPR120 in the context of n-3 vs. n-6 PUFA-induced atheroprotection.
METHODS
Detailed experimental procedures and methods are reported in the online data
supplement. Methods include generation and validation of LDLrKO/GPR120WT (WT) and
LDLrKO/GPR120KO (KO) mice using BMT, measurements of plasma lipids and lipoproteins,
hepatic lipid content, circulating and splenic monocytosis and neutrophilia, monocyte labeling
and recruitment into aortic intima, aortic and macrophage cholesterol content, and aortic root
atherosclerosis and immunohistochemistry/histology.
STATISTICS
Data are presented as mean ± SEM. All data were tested for significant differences
using 2-way ANOVA (diet vs. genotype) with post-hoc Tukey’s multiple comparison test. All
analyses were performed with Statistica.
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RESULTS
Systemic response to atherogenic diets (AD) enriched in n-3 and n-6 PUFAs
Eight week (wk) old female LDLrKO mice were irradiated and then received bone
marrow from male GPR120 WT or KO mice. LDLrKO recipients recovered from radiation/BMT
induced body weight loss within one week and showed similar and positive weight gain during 6
wks of recovery (Supplemental Fig. I A). Transplantation efficiency was ~95% based on
quantification of the ratio of Syr/SOAT2 intensity of the agarose gel scans (Supplemental Fig. I
B) and verification of leukocyte GPR120 alleles using PCR analysis (Supplemental Fig. I C).
Eight wks after BMT, WT and KO mice were switched from chow to one of the four AD
containing PO, BO, EO, or FO for additional 16 wks. Dietary FA compositions showed relative
enrichment of 18 carbon FAs beyond FADS2 in the BO (11.7% 18:3 n-6) and EO (6% 18:4 n-3)
ADs. AD FA compositions are given in Supplemental table I and are similar to those published
previously33 , except that GLA content (2.3% energy) was approximately half that of the
previous study (3.9% energy) because a different source of BO was used. AD feeding over 16
wks resulted in uniform food consumption (~3-4 gm/day/mouse), body weight gain, and terminal
liver, spleen and gonadal adipose/body weight ratios among all groups irrespective of L-
GPR120 expression (Supplemental Figs. II & III). Additionally, L-GPR120 did not affect
percentage (%) red blood cell (RBC) fatty acid (FA) enrichment. Because WT and KO mice fed
the same AD had similar percentage RBC FA enrichment, data from both genotypes were
pooled and are plotted together (Supplemental Fig. IV). We previously demonstrated that
percentage FA enrichment in plasma and liver phospholipids was similar to that in RBCs33.
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L-GPR120 does not affect plasma lipids and cholesterol lipoprotein distribution
Consistent with previous reports, FO, EO and BO reduced total plasma cholesterol (Figure 1 A
and B), plasma triglycerides (Figure 1 C and D), VLDL cholesterol (Figure 2 A and B), LDL
cholesterol (Figure 2 C and D) and raised HDL cholesterol (Figure 2 E and F) relative to PO.
The absence of L-GPR120 had no effect on any of the plasma lipid and lipoprotein
concentrations.
L-GPR120 does not affect hepatosteatosis
Previous reports have shown a phenotype switching (M2 to M1-like) of macrophages in
GPR120 KO vs. WT mice fed high fat diet containing n-3 PUFA or GPR120 selective agonist7, 9.
We determined the extent to which L-GPR120 affects hepatic inflammatory and lipogenic gene
expression, and hepatic lipid content. PO-fed mice had similar hepatic inflammatory gene
expression regardless of L-GPR120 expression (Figure 3A). However, FO, EO and BO-fed
GPR120KO mice had increased M1 (CD11c, MCP-1, TNF-α, IL-1β, and IL-6) and reduced M2
(IL-10, Arginase-1, and iNOS) gene expression compared to WT mice, and increased hepatic
TNF-α and IL-1β protein content (Figure 3 B). FO KO vs. WT mice also had reduced
expression of macrophage marker genes, CD68 and F4/80 (Figure 3A), whereas other diet
groups did not. In general, L-GPR120 expression had no effect on hepatic lipogenic gene
expression (Figure 3C) and hepatic lipid content (Figure 3D), except for FO-fed mice, in which
expression of several lipogenic genes (SREBP-1c, FAS, SCD-1, SREBP-2, HMGCoA synthase,
HMGCoA reductase, and PPARα) was significantly reduced in L-GPR120 KO mice.
Additionally, liver sections of WT and KO mice fed the same AD had similar morphological
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appearance by H&E and CD68 IHC staining (Supplemental Fig. V). Thus, reduced
hepatosteatosis in FO, EO and BO vs. PO-fed mice is independent of L-GPR120 expression
and likely occurs via reduced SREBP1 activation and lipogenic gene expression.
PUFAs attenuate neutrophilia and monocytosis via leukocyte GPR120
Hypercholesterolemia induces leukocytosis, primarily by elevating circulating monocytes
(monocytosis) and neutrophils (neutrophilia), both of which are positively associated with
atherosclerosis in mice and humans 34, 35. The percentage of circulating neutrophils
(CD11b+,CD115-, Ly6G+) was decreased with FO, EO, and BO feeding relative to PO, which
was reversed in the absence of L-GPR120 (Figure 4 A). The spleen is an extramedullary
reservoir for circulating, undifferentiated monocytes, which upon recruitment to a site of injury/
inflammation may differentiate into macrophages, dendritic cells, or other tissue descendants36,
37. Splenic neutrophilia (Figure 4 B) and monocytosis (%CD11b+,CD115+, Ly6G-) (Figure 4C)
were significantly attenuated by dietary FO, EO and BO in mice expressing L-GPR210, but not
those lacking L-GPR120, relative to PO-fed mice. Splenic Ly6Chi monocytosis was also
significantly attenuated in FO, EO and BO-fed WT mice, relative to PO, but not KO mice,
suggesting that L-GPR120 plays a significant role in maintaining a favorable Ly6Clo monocyte
profile in FO, EO and BO-fed mice (Figure 4 D). Despite L-GPR120 expression and dietary
PUFA effects on splenic monocytosis, neither affected the percentage of circulating
monocytosis or Ly6Chi monocytes (Supplemental Fig. VI).
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L-GPR120 attenuates monocyte infiltration into atherosclerotic lesions
GPR120 negatively regulates macrophage chemotaxis ex vivo7. We previously showed that
ADs containing FO, EO and BO, compared to PO, reduce macrophage chemotaxis ex vivo and
result in reduced aortic root CD68+ intimal area in LDLrKO mice33. We hypothesized that
activated L-GPR120 may limit monocyte trafficking into aortic root intima and performed
monocyte-labeling experiments to test our hypothesis. Blood monocytes were phagocytically
labeled with 1µm fluorescent beads using the procedure developed by Randolph and
collegues38, 39. Using this procedure, ~1% of blood leukocytes were labelled with beads (bead+)
and of these, 60-80% were monocytes, resulting in a range of 6-8 % monocyte labeling among
the groups (Supplemental figure VII A). Monocyte recruitment into aortic root intima was
significantly reduced for mice consuming FO, EO and BO, relative to PO, as anticipated;
however, recruitment for all three PUFA groups was equivalent to PO in mice lacking L-GPR120
(Figure 5 A and Supplemental figure VII B). Additionally, splenic Ly6Chi monocytosis
positively correlated with monocyte infiltration into aortic root intima (Figure 5 B).
L-GPR120 expression does not affect atherosclerosis in LDLrKO mice
Aortic root intimal area was significantly lower (500-700 vs. 950-1050 mm2) in FO, EO and BO
fed vs. PO-fed mice (Figure 6 A), as was aortic root intimal macrophage content (CD68+)
(Figure 6 B). L-GPR20 expression did not affect aortic root intimal area; however,
unexpectedly, we observed further reduction in intimal CD68+ content in KO vs. WT mice fed
FO. Intimal collagen content was similar among all groups of mice except for higher values for
WT mice fed FO (Figure 6 C). We hypothesized that paradoxical lowering of CD68+
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macrophage content despite increased monocyte trafficking in KO FO (vs. WT FO) is plausible
if recruited monocytes egress out of the intima and into the lymphatic system undifferentiated or
that the recruited monocytes differentiate into CD11c+ dendritic cells within intima. We
quantified CD11c+ intimal area and found that KO FO mice had increased CD11c+ intimal area
relative to WT FO mice (Figure 6 D). Representative aortic root images of WT and KO mice
corresponding to Fig. 6 A, B, C and D can be found in Supplemental figures VIII, IX, X and XI,
respectively. We also found the number of cleaved caspase-3+ apoptotic cells was similar
among all groups of mice (Supplemental Figure XII). To examine atherosclerosis at another
site, we measured whole aorta FC (Figure 7 A) and CE (Figure 7 B) content, which was
significantly lower in FO, EO and BO fed (~2 fold reduction in FC; ~3-4 fold reduction in CE) vs.
PO-fed mice. In agreement with aortic root intimal area results, L-GPR120 expression had no
impact on this measurement of atherosclerosis, indicating that PUFA-induced atheroprotection
is not significantly affected by L-GPR120 expression.
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DISCUSSION
Whether in vivo L-GPR120 activation by PUFAs is atheroprotective is unknown. In this
study, we determined the extent to which L-GPR120 expression was atheroprotective in the
context of n-3 vs. n-6 PUFA feeding. We hypothesized that L-GPR120 expression contributes to
atheroprotective effects of n-3, but not n-6, PUFAs, since in vitro studies suggest that n-3
PUFAs preferentially activate GPR1207. Our study led to several novel observations. First, we
show that L-GPR120 deletion had minimal effects on plasma lipid concentrations, lipoprotein
cholesterol distribution, hepatic neutral lipid content, aortic root intimal area, and aortic
cholesterol content regardless of dietary fat type fed to the mice. Second, in vivo activation of L-
GPR120 by PUFA-enriched diets limits: a) hepatic macrophage (i.e., Kupffer cell) pro-
inflammatory gene expression, b) splenic and blood neutrophilia, c) splenic Ly6Chi monocytosis,
and d) monocyte recruitment into aortic root atherosclerotic lesions. Third, there was no
distinction between n-3 vs. n-6 PUFAs with regard to in vivo activation of GPR120. These data
suggest that dietary n-3 and n-6 PUFAs, though atheroprotective in LDLrKO mice relative to
PO, have GRP120-independent (i.e., attenuation of hypercholesterolemia, hepatosteatosis, and
aortic atherosclerosis) and -dependent (i.e., attenuation of pro-inflammatory gene expression,
neutrophilia, splenic monocytosis and monocyte trafficking) atheroprotective roles.
We investigated whether GPR120 activation by n-3 PUFAs would reduce atherogenesis
based on its role in attenuating inflammation and insulin resistance in diet-induced obesity7, 9.
GPR120 is highly expressed in macrophages, which are important inflammatory cells in the
pathogenesis of obesity and atherosclerosis7. Mice fed a high fat diet have increased monocyte
recruitment to adipose tissue, adipose tissue macrophage inflammation, and insulin resistance7.
This phenotype was reversed in mice fed a high fat diet in which FO was isocalorically (27% of
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calories) substituted for saturated fat; however, this was not the case for GPR120 KO mice or
mice transplanted with GPR120 KO bone marrow, suggesting that most of the beneficial effect
of FO was due to macrophage GPR120 activation7. Oh et al have shown that activation of
GPR120 results in a beta arrestin 2-mediated internalization of GPR120 and binding to TAB1,
preventing its activation of TAK1 and blunting a common node of inflammatory signaling for Toll-
like receptors, TNFα receptor, and inflammasome activation7. However, another study using a
different GPR120 gene targeting construct showed that reversal of insulin resistance by feeding
a high n-3 PUFA-containing diet was independent of GPR120 expression, suggesting the in vivo
metabolic impact of GPR120 expression is not fully elucidated40.
Central to the pathogenesis of atherosclerosis is lipid-laden macrophages that initiate a
chronic inflammatory state41. Dietary PUFAs are atheroprotective in mice, non-human primates,
and humans11-20; however, the extent to which this atheroprotection is related to GPR120
expression, in general, and macrophage GPR120 expression, in particular, is unknown. Our
results show that dietary PUFA-induced atheroprotection was independent of BM GPR120
expression by several measurements, including aortic root intimal area and whole aorta
cholesterol content. There may be several explanations for this unexpected outcome. First,
inflammation likely plays a minor role in the pathogenesis of atherosclerosis in LDLrKO mice, in
which apoB lipoproteins are elevated in plasma. Past studies from our lab and others have
shown that atherosclerosis is highly associated with plasma VLDL cholesterol concentrations in
LDLrKO mice33, 42. Second, cells outside the BM compartment (i.e., endothelial and smooth
muscle cells) that express GPR120 may play important atheroprotective roles in the context of
dietary PUFA feeding and these would have been missed in our study. Finally, the intake of
dietary PUFAs may not have been sufficient to activate GPR120 in vivo, as our study used
much lower n-3 PUFA enriched diets compared to previous studies7, 40.
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To determine whether diet enrichment of PUFAs was sufficient for in vivo activation of
macrophage GPR120 in our study, we made several measurements of macrophage function.
Mice fed the PUFA-containing diets had decreased percentage of splenic Ly6Chi monocytes and
decreased trafficking of blood monocytes into aortic root atherosclerotic lesions relative to PO-
fed mice and these trends were reversed in mice transplanted with GPR120 KO vs. WT BM.
Blood and splenic neutrophilia was also suppressed in mice transplanted with WT BM and fed
PUFAs, but not PUFA-fed mice transplanted with GPR120 KO BM. In addition, PUFA-fed mice
lacking BM GPR120 had higher hepatic pro-inflammatory gene expression than their WT
counterparts. Since liver has low GPR120 expression4, 8, whereas macrophages have relatively
high expression7, these results suggest that hepatic Kupffer cells, as well as circulating
monocytes and neutrophils, display dietary PUFA-mediated GPR120 activation in vivo. Although
hypercholesterolemia is associated with monocytosis19 , our outcomes were unrelated to
plasma cholesterol concentrations, which were equivalent among all PUFA fed mice regardless
of BM GPR120 expression. Past studies showed in vivo GPR120 activation (i.e., decreased
inflammation, increased insulin sensitivity) at much higher, super physiological intakes of FO7.
As discussed in our previous publication33, the levels of dietary PUFA intake in our study were in
a physiological range that would be achievable for human consumption. Whether diminished
monocytosis and inflammation will occur in humans consuming diets with a similar PUFA
enrichment is unknown. However, adipose tissue GPR120 expression is increased in obese
humans and a GPR120 coding variant that inhibits its activation increases obesity risk in
Europeans. Collectively, our results support the conclusion that sufficient PUFA enrichment
occurred in vivo to activate GPR120, but the anticipated GPR120-mediated reduction in
atherosclerosis was not observed because the plasma lipid lowering effect of the PUFA diets
was overwhelming.
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Our study also demonstrated that the in vivo activation of GPR120 was not specific for n-
3 PUFAs, but was equally effective with dietary n-6 PUFAs. The fatty acid specificity of GPR120
activation has been investigated primarily using in vitro techniques and results are contradictory.
Some report a preference of n-3 PUFAs for GPR120 activation, whereas other studies report
broader fatty acyl specificity. In vivo studies also have yielded conflicting results. Oh et al
showed that a high fat diet enriched in FO reduced glucose intolerance in a GPR120 dependent
manner, whereas Bjursell et al demonstrated that FO containing high fat diet reversed glucose
intolerance in both WT and GPR120 KO mice. The explanation for these disparate outcomes
has been discussed at length by Bjursell et al; possibilities include different gene targeting
constructs, mouse backgrounds, and dietary n-3 PUFA content and length of diet feeding.
Although our primary outcome, which was atherosclerosis progression, was not affected by L-
GPR120 expression, similar to results published by Bjursell et al on glucose intolerance, our
results clearly showed an impact of L-GPR120 on monocyte/macrophage function with both n-3
and n-6 PUFA diets, suggesting the in vivo specificity for L-GPR120 activation is equal for both
classes of PUFAs.
Whole body deletion of GPR120 has led to increased hepatosteatosis in some7, 8, but not
all studies40. In our study, PUFA-containing diets uniformly reduced hepatic neutral lipid content
relative to PO regardless L-GPR120 expression. However, LDLrKO mice transplanted with
GPR120 KO vs. WT BM had increased liver inflammatory gene expression, which we attribute
to Kupffer cells (see above), but only in the PUFA-fed groups. The reason for a PUFA-induced
increase in inflammatory gene expression only in mice lacking L-GPR120 and its relationship to
hepatic lipid content is unknown. We speculate that hepatic Kupffer cell GPR120 expression
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normally suppresses inflammation, perhaps initiated by increased PUFA oxidation, in the
context of PUFA-enriched diets. The increase in hepatic inflammation in PUFA-fed L-GPR120
KO mice may suppress hepatic lipogenesis (ref for increased inflammation decreases hepatic
lipogenesis), resulting in similar hepatic lipid content for L-GPR120 WT and KO mice. The
difference in hepatic lipid content between our study and that of Oh et al may relate to global vs.
BM KO of GPR120 or differences in dietary content of n-3 PUFAs.
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ACKNOWLEDGEMENTS
We gratefully acknowledge Karen Klein (Biomedical Research Services and Administration,
Wake Forest School of Medicine) for editing the manuscript; Xuewei Zhu for help with bone
marrow isolation and retro-orbital injections during monocyte recruitment experiment and Sandy
Chan for help in organ harvesting during terminal necropsies and quantification of aortic root
intimal areas to determine intersubject variability.
SOURCES OF FUNDING
This study was supported by grants from National Institute of Health P50 AT002782 and
R01HL119962 (to JSP).
DISCLOSURES
None.
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FIGURE LEGENDS
Figure 1. L-GPR120 expression does not affect plasma lipids. Irradiated LDLrKO mice
received bone marrow from WT or GPR120 KO donors and were fed ADs (PO, FO, EO or BO)
for 16 wks. Fasting (4h) plasma total cholesterol (TC) (A) and triglyceride (TG) (B)
concentrations were measured by enzymatic assays. Plasma TC (C) and TG (D) concentrations
were integrated over time (0-14 weeks) and expressed as area under the curve (AUC). Mean ±
SEM, n=13-15/diet. Groups with different letters are significantly different (p<0.05) by two-way
ANOVA and Tukey’s post-hoc analysis.
Figure 2. L-GPR120 does not affect plasma lipoprotein cholesterol distribution. Fasted
(4h) plasma was harvested from AD fed WT and KO mice and fractionated by FPLC to
determine cholesterol distribution among VLDL (A), LDL (B) and HDL (C) fractions. Plasma
VLDL-C (D), LDL-C (E) and HDL-C (F) concentrations were integrated over time (0-14 weeks)
and expressed as area under the curve (AUC). Mean ± SEM, n=3 equal volume pooled samples
from 4 mice/group. Groups with different letters are significantly different (p<0.05) by two-way
ANOVA and Tukey’s post-hoc analysis.
Figure 3. Effect of L-GPR120 expression on hepatic gene expression and lipid content.
Livers were harvested from 16 week AD fed WT and KO mice to measure lipid content and
mRNA abundance. Macrophage gene expression (A), lipogenic gene expression (B), and
hepatic neutral lipid content (C) Neutral lipid= CE+TG. Liver was lipid extracted and TC, FC, and
TG were quantified by enzymatic assay. CE mass was calculated as (TC-FC)*1.67 and
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normalized to protein content. Mean ± SEM, n=6. Groups with different letters are significantly
different (p<0.05) by two-way ANOVA and Tukey’s post-hoc analysis.
Figure 4. Effect of L-GPR120 on neutrophilia and monocytosis. Blood and spleens were
harvested from 16 week AD fed WT and KO mice to measure the distribution of neutrophils and
monocytes, including Ly6Chi monocytes, using flow cytometry. Percentage blood neutrophils
(A), percentage splenic neutrophils (B), percentage splenic monocytes (C) and percentage
splenic Ly6Chi monocytes (D). Mean ± SEM, n=12-15/group. Groups with different letters are
significantly different (p<0.05) by two-way ANOVA and Tukey’s post-hoc analysis.
Figure 5. Effect of L-GPR120 on monocyte recruitment to aortic root intima. Blood
monocytes were labeled using 1-µm Fluoresbrite microparticles in WT and KO mice fed AD for
15 weeks. Percentage of FITC bead-labeled blood monocytes was measured by flow cytometry
24 hr after injection. Five days later, the number of FITC+ beads was quantified in the intimal
area of 8 µm thick aortic root sections and normalized by the percentage of FITC+ blood
monocytes to obtain normalized frequency of monocytes recruited into aortic intima (A). Mean ±
SEM, n=6-7/group. Groups with different letters are significantly different (p<0.05) by two-way
ANOVA and Tukey’s post-hoc analysis. Percentage of splenic Ly6Chi monocytes was plotted vs.
monocyte recruitment into aortic intima (B). Each data point represents individual animal and
symbols represent their respective groups, n=6-7/group. The line of best fit, determined by
linear regression analysis, is also plotted.
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Figure 6. Histological quantification of aortic root atherosclerotic lesions. WT and KO
mice were fed AD for 16 weeks. At necropsy, hearts were frozen in OCT, and aortic roots were
serially sectioned. Quantification of aortic root Oil Red O positive intimal area (lesion area) (A).
Quantification of percentage aortic root lesional area occupied by CD68+ cells (macrophages)
(B). Quantification of percentage aortic root lesional area occupied by Sirius red positive
collagen fibers under plane polarized light (C). Quantification of percentage aortic root lesional
area occupied by CD11c+ cells (D). Each point represents the average area from 8-10 sections
for an individual mouse. Horizontal lines denote the mean for each diet group. Representative
images for each parameter are depicted in Supplemental Figures VIII, IX, X and XI. n=6-
8/group. Groups with different letters are significantly different (p<0.05) by two-way ANOVA and
Tukey’s post-hoc analysis.
Figure 7. Effect of L-GPR120 expression on aortic cholesterol content. Aortas were
harvested from 16 week AD fed WT and KO mice for atherosclerosis quantification. Aortas were
lipid extracted for measurement of total cholesterol and free cholesterol (FC) content using gas-
liquid chromatography. Cholesterol ester (CE) content was calculated as (TC-FC) x1.67. Aortic
FC content (A). Aortic CE content (B). n=12-14/group, each data point represents an individual
mouse aorta. Horizontal lines denote the mean for each diet group. Groups with different letters
are significantly different (p<0.05) by two-way ANOVA and Tukey’s post-hoc analysis.
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Novelty and Significance
What Is Known?
GPR120 is an n-3 fatty acid receptor mediating potent anti-inflammatory and insulin-
sensitizing effects
Botanical oils enriched in n-6 and n-3 fatty acid products of FADS2 are equally effective
in preventing atherosclerosis and hepatosteatosis in mice
What New Information Does This Article Contribute?
L-GPR120 is neither necessary nor sufficient for atheroprotection in LDLrKO mice fed n-
6 or n-3 PUFA-enriched diets
Physiological levels of dietary PUFA intake in LDLrKO were sufficient for in vivo
activation of L-GPR120, leading to attenuated neutrophilia, splenic monocytosis, and
monocyte recruitment into aortic intima
GPR120 activation in vivo was not n-3 PUFA selective; n-6 PUFAs also activate
GPR120
Deletion of GPR120 expression in hepatic Kupffer cells results in increased inflammation
in the context of increased dietary consumption of PUFAs independent of hepatic neutral
lipid content
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CHAPTER III: Supplemental Material
Supplemental Methods and Results
Dietary oils
The seed oil of Borago officinalis L., a member of the Boraginaceae family, was generously
donated by Nordic Naturals (Watsonville, CA, USA). The seed oil of Echium plantagineum L., a
member of the Boraginaceae family was a generous gift from Croda Europe Ltd. (Leek,
Staffordshire, UK). All oils were authenticated by the Wake Forest University Center for
Botanical Lipids and Inflammatory Disease Prevention. A certificate of analysis is on file for
reference along with retention samples deposited at the Wake Forest School of Medicine. The
seed oil of palm, Elaeis guineensis Jacq., a member of the Arecaceae family, was purchased
from Shay and Company (Portland, OR, USA). A certificate of analysis is on file for reference
along with retention samples deposited at the Wake Forest School of Medicine. The fish oil
source was Brevoortia tyrannis Latrobe, a member of the Clupeidae family, was manufactured
and generously provided by Omega Protein (Houston,TX, USA) with a report of analysis on file
for reference.
Animals and Atherogenic Diets
Female LDLrKO (C57BL/6 background) mice (5-6 weeks of age) were purchased from The
Jackson Laboratory (Bar Harbor, Maine, USA). Mice were housed in a specific pathogen-free
facility on a 12h light/dark cycle. Mice were allowed to acclimate to in-house animal resource
facilities for 1-2 weeks. At 8 weeks of age, mice received bone marrow from either WT or KO
GPR120 male mice (see below). After ~6 weeks of recovery from bone marrow transplantation
(BMT) and at 13-14 weeks age, mice were randomly assigned to one of four atherogenic diet
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(AD) groups (n=15/ diet group) containing 10% calories as PO and 0.2% cholesterol,
supplemented with an additional 10% of calories as: 1) PO, 2) Borage oil (BO; 18:3 n-6
enriched), 3) Echium Oil (18:4 n-3 enriched), or 4) Fish oil (FO; 20:5 n-3 and 22:6 n-3 enriched)
for additional 16 weeks. All protocols and procedures were approved by the Institutional Animal
Care and Use Committee. ADs were prepared by the diet kitchen in the Department of
Pathology at Wake Forest School of Medicine as previously described 1. Detailed composition
and quality control data for similar ADs has been published2, 3
Bone marrow transplantation
BM cells were harvested from cleaned femurs and tibias of male GPR120 WT and GR120 KO
mice and re-suspended in serum-free RPMI 1640 medium. Female LDLr-/- recipient mice
(Jackson Laboratories) were fasted overnight and received a sublethal dose of radiation (900
rads) 4h prior to BM injection. BM cells (~7 × 10 6/ mouse) were injected into the retro-orbital
venous plexus of anesthetized recipient mice. Recipient mice received autoclaved, acidified (pH
2.7) water supplemented with 100 mg/l neomycin and 10 mg/l polymyxin B sulfate 3 days before
and 2 weeks after the transplantation. Mice were then given autoclaved acidified water until the
end of the study as described previously 4.
Repopulation of blood leukocytes by transplanted GPR120 WT or KO hematopoietic stem cells
in female LDLrKO recipients was evaluated after 6 weeks of recovery by determining
percentage expression of the Y-chromosome-associated sex-determining region Y gene (Sry) in
genomic DNA obtained from white blood cells. Additionally, white blood cell GPR120 WT and
KO genotype was also confirmed using PCR. Briefly, genomic DNA from whole blood was
extracted with a Wizard Genomic DNA Purification kit (Promega). Sry was amplified by PCR to
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a linear amplification phase under the following conditions: 95°C for 3 min followed by 30 cycles
of 94°C for 30 s, 61°C for 1 min, and 72°C for 1 min. Acyl-CoA: cholesterol acetyltransferase 2
(ACAT2) was amplified by PCR as an internal control. The PCR products were separated on
0.8% agarose gels and visualized with ethidium bromide. A series of male and female genomic
DNA mixtures (100, 50, 25, and 0% male DNA) were made and used to construct a standard
curve of male:female DNA ratio vs. Sry:ACAT2 ratio, determined by quantification of density of
PCR bands. Male genomic DNA in the whole blood samples of transplanted mice was
estimated using the standard curve.
Body weight gain and organ weights
Mice were weighed one day prior to and daily for 3 consecutive days after BMT and then on a
weekly basis for 6 weeks until they were started on the ADs. During 16 weeks of AD feeding,
mice were weighed every 2-4 weeks. At necropsy, terminal body weights were recorded after 4
hours of fasting. Mice were then anesthetized using ketamine-xylazine (intramuscularly) and
perfused through the left ventricle using cold PBS at the rate of ~3ml/minute for 3-4 minutes
prior to organ harvesting. After perfusion, liver, spleen, and adipose wet weight was measured
and normalized to terminal body weight.
Fatty acid analysis
Lipid extraction of lyophilized diets and RBCs was performed using the Bligh-Dyer method 5.
The lipid extracts were trans-methylated using boron trifluoride (BF3), and percentage fatty acid
composition was quantified by gas–liquid chromatography as described previously 1.
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Plasma lipid and lipoprotein analysis
Blood was collected from 4h-fasted mice by tail bleeding and plasma was isolated by low speed
centrifugation. Plasma total and free cholesterol (Wako), and triglyceride (Roche)
concentrations were determined using enzymatic assays as described earlier 6 at baseline and
after 2, 4, 8, and 16 weeks of AD feeding. Cholesteryl ester content was calculated as (TC-FC)
x 1.67; the multiplication by 1.67 corrects for loss of fatty acid during the cholesterol esterase
step of the assay. Data were expressed as area under the curve (AUC), which integrates
plasma lipid concentrations over the 16 weeks of AD feeding, representing a time average
estimate of hypercholesterolemia throughout atherosclerosis progression. Plasma lipoprotein
cholesterol mass distribution was determined using fast protein liquid chromatography (FPLC)
fractionation on a Superose 6 10/300 column (GE Healthcare). Three equal volume plasma
samples from 5 mice/group were pooled and subjected to FPLC fractionation and cholesterol
quantification at baseline (chow diet) and at 2, 4, 8, 12 and 16 weeks of AD feeding. VLDL, LDL
and HDL cholesterol concentrations were determined and then expressed as AUC.
Liver Lipids
Livers were harvested at necropsy, flash frozen in liquid N2, and stored at -80 C. Liver lipids
were quantified using a detergent-based enzymatic assay as described earlier 7.
Flow cytometry
Peripheral blood was obtained for circulating leukocyte analysis. For splenic cells, tissue was
digested with an enzyme cocktail as published8. The cell suspension was subsequently passed
through a 70-µm nylon cell strainer (BD Falcon). Red blood cells were removed from flow
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cytometry preparations by treatment with ACK lysing buffer (Gibco). The remaining white blood
cells were incubated with the following mAbs: CD11b-APC-Cy7, clone:M1/70 (BioLegend);
CD115-APC, clone:AFS98; CD62L-PerCP-Cy5.5, clone:MEL-14 (eBiosciences); Ly6G-PE,
clone:1A8; CD11c-PE-Cy7, clone:HL3; and Ly6C-FITC or PE clone:AL-21 (BD Pharmingen).
Data were acquired on a BD FACSCanto II instrument (BD Biosciences) and analyzed using
FacsDiva software (BD Biosciences).
Monocyte labeling
Monocytes were labeled following the Gr-1lo method as described by Tacke et al. 9, 10. One-µm
Fluoresbrite Yellow Green microspheres (2.5% solids [wt/vol], Polysciences, Inc., Warrington,
PA) were diluted in PBS (1:4) and retroorbitally injected into anesthetized mice. Blood was
harvest from mice 24 hours after labeling for evaluation of bead-containing monocytes. The
method labels predominantly Ly6Clo monocytes 9.
Quantification of monocyte recruitment
Monocyte recruitment was evaluated as reported previously11. Briefly, aortic root sections were
taken as described below and the number of beads per section in atherosclerotic lesions was
counted manually at 20x magnification. A total of 8 sections representing the length of the aortic
root were analyzed per mouse. Because of slight variations in monocyte labeling (6-8%) among
animals, data were normalized for individual mice based on the percentage of blood monocytes
labeled. This adjustment gave a normalized value, which represented the actual number of
monocytes entering the lesion (i.e. normalized frequency).
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Quantification of aortic root lesion area
Aortic root atherosclerosis was assessed according to the method of Daugherty et. al12. Aortic
roots were embedded in Optimal Cutting Temperature (Tissuetek) media in a plastic mold,
frozen, and cut at 8 μm intervals. Sections were collected from the aortic root moving toward the
apex of the heart and sequentially placed on 6 slides, such that each slide had sections 48 μm
apart. The sections were fixed in 10% buffered formalin, stained in 0.5% Oil Red O for 25
minutes and counterstained with hematoxylin. Stained sections were photographed with a Nikon
DigitalSight DSFi1 camera and quantified using NIS Elements (Nikon) software. Intimal area
measurements were obtained with ImagePro 6.2 software (Media Cybernetics Inc., Rockville,
Md.). We defined intimal area as the lesion area between the internal elastic lamina and the
luminal surface of the aorta. Eight-10 aortic root sections were quantified from 6-8 mice/group.
The mean coefficient of variation for these measurements from individual mice on atherogenic
diets was 15.0%.
Sirius red staining and quantification
Fresh frozen aortic root sections were fixed (10% buffered formalin, 10 minutes), washed (DI
water, 5 minutes) and dehydrated in 50, 75 and 80 % ethanol, sequentially, for 1 minute each.
Sections were stained using hematoxylin, and then stained in 1% Sirius red dye (Sigma Aldrich
#365548) in saturated solution of picric acid (1.3 % picric acid in H2O Sigma Aldrich # P6744)
for 60 minutes. After staining with Sirius red, sections were washed in acidified water (5%
glacial acetic acid) for 5 minutes and dehydrated in increasing ethanol concentrations, cleaned
in xylene and cover slipped using xylene based mounting media. Images were acquired under
plane polarized light using a Nikon DigitalSight DSFi1 camera fitted with T-A2-DIC analyzer
(Nikon # MEN51921) and T-P2 DIC polarizer (Nikon # MEN51951).
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Immunohistochemistry
Aortic root cross-sections and liver sections were fixed and incubated with anti-CD68 antibody
(AbDSerotec, clone FA11), anti-CD11c antibody (BD Pharminogen, clone HL3) or anti-cleaved
caspase -3 antibody (Abcam, polyclonal # ab2302) (1:200, overnight) as described previously11.
Analysis of aortic cholesterol content
Aortic total, free and cholesteryl ester content was measured as described earlier 11.
Statistical analyses
Data are presented as mean ± SEM. All data were tested for significant differences using 2-way
ANOVA with post-hoc Tukey’s multiple comparison test. All the analyses were performed with
Statistica.
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Supplementary Table 1
Table 1: Atherogenic Diet (AD) percentage fatty acid composition (%FA) and percentage
total energy equivalence (% EE) of individual fatty acid
Palm oil (PO) Fish oil (FO) Echium oil (EO Borage oil (BO)
Fatty Acid % FA % FA % EE % EE % FA % EE % FA % EE
Palmitic Acid
(C16:0)
43.2 8.89 30.6 6.89 25.6 5.27 26.0 5.20
Palmitoleic Acid
(C16:1)
0.37 0.08 4.2 0.95 0.4 0.02 0.4 0.08
Stearic Acid
(C18:0)
4.5 0.93 4.5 1.01 4.1 0.84 3.5 0.7
Oleic Acid
(C18:1 n-9)
37.3 7.68 24.9 5.61 26.1 5.37 25.1 5.02
Linoleic Acid
(C18:2 n-6), LA
11.1 2.29 8.2 1.85 15.4 3.17 25.0 5.0
α-Linolenic Acid
(C18:3 n-3), ALA
0.37 0.08 1.0 0.23 13.8 2.84 0.4 0.08
γ-Linolenic Acid
(C18:3 n-6), GLA
5.0 1.03 11.40 2.28
Stearidonic Acid
(C18:4 n-3), SDA
1.5 0.34 6.0 1.23 0.2 0.04
Euric acid
(C22:1 n-9)
0.2 0.04 0.9 0.18
Eicosapentaenoic
Acid (C20:5 n-3),
EPA
0.1 0.02 6.9 1.55 0.3 0.06 0.3 0.06
Docasahexaenoic
Acid (C22:6 n-3),
DHA
0.2 0.04 7 1.58 0.3 0.06 0.3 0.06
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ADs contained 0.2% cholesterol + 10% calories as Palm Oil (PO) + 10% calories as: PO,
Borage oil (BO), Echium oil (EO) or Fish oil (FO). Percent (%) fatty acid composition was
measured using gas-liquid chromatography. Percent total energy equivalence (% EE) for
individual fatty acid is calculated using total energy derived from fatty acids (i.e. 20%) / diet and
% fatty acid composition of respective diet.
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Supplementary Figure Legends
Supplementary Fig. I. Bone marrow transplantation (BMT) efficiency. Irradiated LDLrKO
mice received bone marrow from WT or GPR120 KO donors. After 6 weeks of recovery, mice
were bled to isolate genomic DNA from circulating leukocytes and were subjected to genotyping
for male gene “SRY” to verify BMT efficiency and GPR120 deletion. Body weight prior to and
after BMT (A), expression of SRY and SOAT22 (positive control) in leukocyte genomic DNA
obtained from female LDLrKO recipients (B), and GPR120 genotyping for WT and KO alleles in
leukocyte genomic DNA isolated from WT or KO BM recipients (C).
Supplementary Fig. II. Body weight gain and terminal body weights. Irradiated LDLrKO
mice received bone marrow from WT or GPR120 KO donors, were fed the indicated ADs for 16
weeks, and body weight gain was measured periodically (mean ± SEM; n=12-15/diet group) (A).
At 16 weeks, mice were euthanized and terminal body weights were noted. Each point
represents terminal body weight for an individual mouse. Horizontal lines denote the mean for
each diet group, n=12-15/diet group (B). No significant differences were found by two-way
ANOVA.
Supplementary Fig. III. Terminal organ/body weight ratios Irradiated LDLrKO mice received
bone marrow from WT or GPR120 KO donors and were fed ADs (PO, FO, EO or BO) for 16
weeks. Mice were then euthanized and liver (A), spleen (B), and gonadal adipose tissue (C) wet
weights were measured and normalized to terminal body weight., Each point represents
terminal body weight/ organ weigh ratio for an individual mouse,. Horizontal lines denote the
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mean for each diet group, n=12-15/diet group. No significant differences were found by two-way
ANOVA.
Supplementary Fig. IV. RBC fatty acid (FA) composition. RBCs were harvested from WT
and KO mice consuming the indicated AD at baseline (chow diet) and after 6 weeks of AD
feeding; fatty acid percentage distribution was measured as described in the methods. Data for
individual fatty acids are expressed as percentage composition of total fatty acids. An equal
volume pool of RBCs from 4 mice/diet group/genotype was used for analysis. Since no
significant differences were detected between WT and KO mice fed the same AD, WT and KO
data were pooled and plotted for mice fed the same AD. Mean ± SEM, n=8. Groups with
different letters are significantly different (p<0.05) by one-way ANOVA and Tukey’s post-hoc
analysis.
Supplementary Fig. V. Liver Histology. Livers were harvested from 16 week AD-fed WT and
KO mice, formalin fixed, sectioned (8um thick), and stained for H&E and CD68 using
immunohistochemistry (IHC). Representative H&E stained liver sections (A) and CD68 IHC
images (B) from WT and KO mice fed each AD. Qualitative microscopic observation did not
reveal any histological differences between WT and KO mice fed the same AD.
Supplementary Fig. VI. Effect of L-GPR120 on circulating monocytosis. Irradiated LDLrKO
mice received bone marrow from WT or GPR120 KO donors and were fed ADs (PO, FO, EO or
BO) for 16 weeks before blood was harvested to measure the distribution of monocytes by flow
cytometry. Monocytes were defined as CD11b+, CD115+, Ly6G- cells. Percentage blood
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monocytes (A) and % blood Ly6Chi monocytes (B). n=12-14/group. Groups with different letters
are significantly different (p<0.05) by two-way ANOVA and Tukey’s post-hoc analysis.
Supplementary Fig. VII. Monocyte recruitment experiment. Irradiated LDLrKO mice received
bone marrow from WT or GPR120 KO donors and were fed ADs (PO, FO, EO or BO) for 16
weeks. Percentage blood monocyte labeling efficiency was measured as % FITC+, CD115+,
Ly6G- cells 24 hours after Fluoresbrite microparticle injection. n=12-14/group. No significant
differences were found by two-way ANOVA (A). 5 days later, the number of FITC+ beads was
quantified in intimal area of serially sectioned 8 µm thick aortic roots using fluorescent
microscope. A minimum of 8-10 sections were analyzed and then averaged for individual aortic
root/ animal. n=6-8 animals/group. Bars with different letters denote statistically significant
difference, p<0.05 (B).
Supplementary Fig. VIII. Representative aortic root atherosclerotic lesions stained for Oil-
red-O. Irradiated LDLrKO mice received bone marrow from WT or GPR120 KO donors and
were fed ADs (PO, FO, EO or BO) for 16 weeks. At necropsy, hearts were frozen in OCT, and
aortic roots (8 µm thick) were serially sectioned and stained using Oil Red O. Representative
images of aortic root Oil Red O positive intimal area (lesion area) of individual WT and KO mice
for each diet group.
Supplementary Fig. IX. Representative aortic root atherosclerotic lesions stained for
CD68/ macrophages. Irradiated LDLrKO mice received bone marrow from WT or GPR120 KO
donors and were fed ADs (PO, FO, EO or BO) for 16 weeks. At necropsy, hearts were frozen in
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OCT, and aortic roots (8 µm thick) were serially sectioned and stained using anti-CD68
antibody. Representative images of aortic root CD68+ intimal area (lesion area) of individual WT
and KO mice for each diet group.
Supplementary Fig. X. Representative aortic root atherosclerotic lesions stained for
Sirius red/ collagen. Irradiated LDLrKO mice received bone marrow from WT or GPR120 KO
donors and were fed ADs (PO, FO, EO or BO) for 16 weeks. At necropsy, hearts were frozen in
OCT, and aortic roots (8 µm thick) were serially sectioned and stained using picrosirius red dye.
Representative images of aortic roots obtained under plane polarized light of individual WT and
KO mice fed respective AD.
Supplementary Fig. XI. Representative aortic root atherosclerotic lesions stained for
CD11c/ dendritic cells. Irradiated LDLrKO mice received bone marrow from WT or GPR120
KO donors and were fed ADs (PO, FO, EO or BO) for 16 weeks. At necropsy, hearts were
frozen in OCT, and aortic roots (8 µm thick) were serially sectioned and stained using anti-CD68
antibody. Representative images of aortic root CD68+ intimal area of individual WT and KO
mice fed FO. Mice fed other (than FO) atherogenic diets did not differ from one another (not
depicted).
Supplementary Fig. XII. Representative aortic root atherosclerotic lesions stained for
cleaved-caspase-3/ apoptotic cells. Irradiated LDLrKO mice received bone marrow from WT
or GPR120 KO donors and were fed ADs (PO, FO, EO or BO) for 16 weeks. At necropsy,
hearts were frozen in OCT, and aortic roots (8 µm thick) were serially sectioned and stained
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using anti-cleaved caspase-3 (CC-3) antibody. Representative images of aortic root CC-3+
intimal area of individual WT and KO mice fed FO. Mice fed other (than FO) atherogenic diets
did not differ from one another (not depicted).
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Supplementary Figures
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Reference List
1. Rudel LL, Kelley K, Sawyer JK, Shah R and Wilson MD. Dietary monounsaturated fatty
acids promote aortic atherosclerosis in LDL receptor-null, human ApoB100-overexpressing
transgenic mice. Arteriosclerosis, thrombosis, and vascular biology. 1998;18:1818-27.
2. Shewale SV, Boudyguina E, Zhu X, Shen L, Hutchins PM, Barkley RM, Murphy RC and
Parks JS. Botanical oils enriched in n-6 and n-3 fatty acid products of FADS2 are equally
effective in preventing atherosclerosis and hepatosteatosis in mice. Journal of lipid research.
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3. Zhang P, Boudyguina E, Wilson MD, Gebre AK and Parks JS. Echium oil reduces
plasma lipids and hepatic lipogenic gene expression in apoB100-only LDL receptor knockout
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4. Rong S, Cao Q, Liu M, Seo J, Jia L, Boudyguina E, Gebre AK, Colvin PL, Smith TL,
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plasma and hepatic lipid levels and exacerbates atherosclerosis. Journal of lipid research.
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6. Zhu X, Lee JY, Timmins JM, Brown JM, Boudyguina E, Mulya A, Gebre AK, Willingham
MC, Hiltbold EM, Mishra N, Maeda N and Parks JS. Increased cellular free cholesterol in
macrophage-specific Abca1 knock-out mice enhances pro-inflammatory response of
macrophages. The Journal of biological chemistry. 2008;283:22930-41.
7. Carr TP, Andresen CJ and Rudel LL. Enzymatic determination of triglyceride, free
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8. Galkina E, Kadl A, Sanders J, Varughese D, Sarembock IJ and Ley K. Lymphocyte
recruitment into the aortic wall before and during development of atherosclerosis is partially L-
selectin dependent. J Exp Med. 2006;203:1273-82.
9. Tacke F, Ginhoux F, Jakubzick C, van Rooijen N, Merad M and Randolph GJ. Immature
monocytes acquire antigens from other cells in the bone marrow and present them to T cells
after maturing in the periphery. J Exp Med. 2006;203:583-97.
10. Tacke F, Alvarez D, Kaplan TJ, Jakubzick C, Spanbroek R, Llodra J, Garin A, Liu J,
Mack M, van Rooijen N, Lira SA, Habenicht AJ and Randolph GJ. Monocyte subsets
differentially employ CCR2, CCR5, and CX3CR1 to accumulate within atherosclerotic plaques. J
Clin Invest. 2007;117:185-94.
11. Brown AL, Zhu X, Rong S, Shewale S, Seo J, Boudyguina E, Gebre AK, Alexander-
Miller MA and Parks JS. Omega-3 fatty acids ameliorate atherosclerosis by favorably altering
monocyte subsets and limiting monocyte recruitment to aortic lesions. Arteriosclerosis,
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12. Daugherty A and Whitman SC. Quantification of atherosclerosis in mice. Methods in
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CHAPTER IV
DISCUSSION
Swapnil V. Shewale
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DISCUSSION
Chronic inflammation, primarily mediated via intimal macrophage accumulation and
activation, plays a critical role in atherosclerotic lesion progression13. Dietary PUFAs impart
atheroprotection in mice, non-human primates and humans11, 14-22. However, the extent to which
this atheroprotection is imparted via attenuation of macrophage inflammation, particularly via
GPR120, and whether activation of leukocyte/ macrophage GPR120 affects lipid-lipoprotein
metabolism was unknown.
We used a dietary approach to achieve efficient in vivo enrichment of cell membranes
and plasma lipoproteins in >18 carbon n-3 or n-6 PUFAs by identifying botanical oils relatively
enriched in n-3 or n-6 PUFAs beyond rate limiting FADS2 enzyme. Using this approach, we
previously showed that an atherogenic diet containing EO, which is relatively enriched in
stearidonic acid (18:4 n-3), the immediate product of FADS2-mediated desaturation of 18:3 n-3,
effectively enriches plasma and tissue lipids in the anti-inflammatory PUFA 20:5 n-3 and was as
atheroprotective as dietary fish oil (FO) compared to the saturated/monounsaturated fatty acid
enriched palm oil (PO)11, 23. However, whether a similar strategy of dietary enrichment in FADS-
2 n-6 products would lead to atheroprotection is unknown. To address this gap in knowledge,
we tested the hypothesis that dietary borage oil (BO), enriched in the FADS-2 product 18:3 n-6,
would not be as atheroprotective as EO, due to in vivo conversion of 18:3 n-6 to 20:4 n-6, a pro-
inflammatory eicosanoid precursor.
Contrary to our hypothesis, we found that BO and EO equally attenuated
atherosclerosis and hepatic neutral lipid accumulation relative to PO. The reduction in
atherosclerosis resulting from BO feeding appeared due to plasma VLDL-c reduction as well as
attenuation of macrophage inflammation and chemotaxis despite significant enrichment of
plasma and liver lipids in 20:4 n-6, a pro-inflammatory eicosanoid precursor (Chapter II).
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However, BO vs. EO feeding did not result in increased 20:4 n-6 derived eicosanoid release in
LPS-stimulated peritoneal macrophage cultured media. In fact, we found that 12/15
lipooxygenase-derived oxidized 18:2 n-6 metabolites, 9 and 13 HODE, were predominantly
secreted over 20:4 n-6-derived eicosanoids. Since oxidized cholesteryl ester (ox-CE) species
derived from 18:2 n-6 have been detected in advanced human atheromas24, we determined
whether ox-CE species may contribute to athero-progression in mouse aortas as well. We found
that mouse aortas were devoid of ox-CE’s, implying a minimal role of ox-CE species in mouse
atherosclerosis.
BO reduced hepatic neutral lipid accumulation in similar fashion to that of EO and FO.
This is a novel observation since diets enriched in 18:2 n-6 do not reduce hepatic neutral lipid
content25, 26. Reduced nuclear SREBP-1 content is likely the key determinant of reduced
hepatic neutral lipid content in BO, EO and FO-fed mice. Multiple mechanisms have been
proposed that may explain reduced nuclear SREBP-1 content including reduced mRNA
expression27, mRNA decay28, proteosomal degradation and inactivation by LXR agonists29.
However, understanding of detailed mechanisms regarding how PUFAs inhibit proteolytic
cleavage of SREBP-1 is limited. Additionally, BO, EO and FO induced hepato-protection, in
part, is mediated via reduction in monounsaturated cholesteryl ester formation via SOAT230.
SOAT2 deletion, similar to BO, EO and FO diet feeding, is atheroprotective and attenuates
hepatic neutral lipid content 31 32. The reduction in hepatic neutral lipid accumulation in BO-fed
mice is despite elevated hepatic TG secretion and plasma TG levels relative to EO and FO. This
paradox is likely due to the fact that quantitatively, only a fraction of hepatic TG is mobilized for
secretion in VLDL; therefore, a large decrease in hepatic TG content does not necessarily result
in decreased hepatic VLDL TG secretion. For example, SCD-1 silencing using anti-sense
oligonucleotide results in a 90% reduction in hepatic TG content, but does not affect hepatic
TG secretion compared to mice treated with a non-targeting anti-sense oligonucleotide33,
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suggesting that a secretory pool of TG may be regulated differently than the bulk TG storage
pool in hepatocytes. Hence, collectively our results show that BO and EO-enriched FADS2 fatty
acid products 18:4 n-3 and 18:3 n-6 PUFAs are efficiently converted to longer chain ≥ 20 carbon
PUFAs, resulting in membrane and plasma lipid enrichment in 20:5 n-3 and 20:4 n-6,
respectively, which in turn, reduces hepatic steatosis and aortic atherosclerosis.
In addition to reduced hepatic neural lipid content, BO, EO and FO fed mice also had
reduced expression of the macrophage marker, CD68, and a chemokine, MCP-1, in liver. We
also observed decreased aortic root macrophage (CD68+ cells) content, decreased
macrophage chemotaxis in vitro, and decreased peritoneal macrophage inflammatory response
to LPS in vitro. These observations prompted us to consider the atheroprotective potential of
GPR120, a negative regulator of macrophage inflammation and chemotaxis.
We determined the atheroprotective potential of GPR120, which is an n-3 fatty acid
receptor mediating potent anti-inflammatory and insulin-sensitizing effects34, 35. Since its
deorphanization, GPR120 has been described as a physiological mediator of long chain
PUFAs36 and as the physiological mediatory of n-3 PUFAs37. GPR120 activation has been
implicated in energy homeostasis, regulation of obesity, insulin sensitivity and chronic
inflammation34-36, 38 . However, whether leukocyte GPR120 (L-GPR120) activation by n-3 vs. n-6
PUFA is atheroprotective in vivo was unknown. We hypothesized that dietary n-3 PUFAs would
lead to greater activation of GPR120 and less inflammation than n-6 PUFAs, since in vivo
activation of GPR120 by n-3 PUFAs has been shown to reverse chronic inflammation and
glucose intolerance resulting from high fat diet feeding (ref). Additionally, GPR120 is highly
expressed in macrophages and bone marrow transplantation studies indicate that most of the
beneficial effects of n-3 PUFA are mediated via macrophage GPR12037. GPR120 activation
blunts the activation of TAK137, a common node of Toll-like receptor mediated pro-inflammatory
signaling and NLRP3 inflammasome activation 39.
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Our study on whether L-GPR120 activation is atheroprotective led to several novel
observations. We report that GPR120 activation in vivo was not n-3 PUFA selective; n-6 PUFAs
also activated GPR120. In our studies, physiological levels of dietary PUFA intake in LDLrKO
were sufficient for in vivo activation of L-GPR120, leading to attenuated neutrophilia, splenic
monocytosis, and monocyte recruitment into aortic intima, whereas deletion of L-GPR120
expression in hepatic Kupffer cells resulted in increased inflammation in the context of
increased dietary consumption of PUFAs, independent of hepatic neutral lipid content (Chapter
III). Although, hypercholesterolemia is known to induce monocytosis, in our studies, L-GPR120
activation attenuated monocytosis and neutrophilia independent of plasma cholesterol content
which was equivalent among all PUFA fed groups. Additionally, L-GPR120 deletion had minimal
impact on plasma lipid concentration, lipoprotein cholesterol distribution, and aortic root intimal
area and aortic cholesterol content. Hence, we conclude that L-GPR120 is neither sufficient nor
required to impart significant atheroprotection or hepatoprotection in LDLrKO mice fed n-3 or n-
6 PUFA-enriched diets.
Contradictory results exist regarding specificity of GPR120 activation by n-3 PUFAs and
its influence on hepatic steatosis34, 35 40. Our study, indicates that either class of PUFAs may
activate GPR120 in vivo. To our knowledge, other studies have not investigated the possibility
of GPR120 activation by FADS2 derived PUFAs although Initial studies had identified: α-linoleic
acid and γ-linoleic acid as high potency fatty acid activators of GPR12035. In a recent study,
DHA, oleic, palmitoleic, palmitic acid were shown to activate GPR120; however n-6 PUFA
including linoleic, γ-linoleic acid and AA were not reported/tested 41.
Additionally, in our study L-GPR120 deletion did not significantly affect hepatic neutral
lipid content, in contrast to other studies using whole body GPR120 KO mice 34, 35, 37. In studies
using whole body GPR120 KO (GPR120 KO) mice, a super-physiological fish oil (27% w/w)
supplementation was fed35, 37. Additionally, only GPR120 KO mice bred on a mixed
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129Sv/C57BL/6 background, in contrast to GPR120 KO mice bred on a pure C57BL/6N
background show increased hepatic neutral lipid content40. In summary, possible explanations
for above discrepancies include different gene targeting constructs, mouse backgrounds, and
dietary n-3 PUFA content and length of diet feeding.
Our results regarding effects of L-GPR120 deletion on monocytosis and neutrophilia
and hepatic Kupffer cell inflammation support the conclusion that sufficient PUFA enrichment
occurred in vivo to activate GPR120, but the anticipated GPR120-mediated reduction in
atherosclerosis was not observed. Factors that may have contributed to above results
regarding L-GPR120 deletion include activation of L-GPR120 in non-bone marrow derived cells
that express GPR120 and may impart an atheroprotective effect. For example, a study showed
GPR120 activation in perivascular adipose tissue (PVAT) by endogenously synthesized n-3
PUFAs in fat-1 transgenic mice protected from femoral arterial thrombosis, neointimal
hyperplasia and vascular inflammation 42. Secondly, in LDLrKO mice, elevated cholesterol
content in ApoB containing lipoproteins, particularly VLDL, rather than increased macrophage
inflammation, is likely the determining factor in pathogenesis of aortic root atherosclerosis43.
As described in Chapter II, the levels of dietary PUFA intake in our study were in a
physiological range that would be achievable for human consumption. Whether similar effects
regarding macrophage inflammation will occur in humans consuming diets with a similar PUFA
enrichment is unknown.
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Implications for human health
A meta-analysis of 13 cohort studies (involving 310,602 individuals and 12,479 coronary
heart disease events) indicate that increased consumption of dietary n-6 PUFAs, particularly LA
is potentially atheroprotective in the general population. A 5% increase in energy intake from LA
is associated with a 10% and 13% lower risk of coronary heart disease events and deaths,
respectively44. However, populations harbouring a single nucleotide polymorphism (rs174537) in
the FADS1/2 gene cluster have a small, but significant, enrichment in plasma AA levels and
show increased production of LTB4 and 5-hydroxyeicosatetraenoic acids in zymosan- stimulated
blood45, 46. Thus, individuals harboring genetic FADS polymorphisms (rs174537) may be
hypersensitive to LA and GLA-enriched diets. However, whether this potential
hyperresponsiveness due to rs174537 affects coronary heart disease risk is unknown.
Additionally, a non-synonymous mutation (p.R270H) inhibits GPR120 signaling activity, resulting
in increased risk of obesity in European populations35, indicating that GPR120 activation by
PUFAs has implications for metabolic disorders. Whether the p.R270H mutation affects
coronary heart disease risk is currently unknown. However, since PUFAs are potential GPR120
ligands and known to reduce coronary heart disease risk, it may be worth determining coronary
heart disease risk in individuals with the p.R270H mutation.
Overall Conclusion
Dietary n-3 and n-6 PUFAs regulate multiple pathways involved in lipid metabolism
and macrophage inflammation. Notably FADS2 derived n-6 PUFAs are as atheroprotective and
hepatoprotective as their n-3 counterparts. Although, some of the effects of dietary PUFAs with
regard to attenuation of macrophage inflammation are primarily mediated via activation of
GPR120, dietary PUFA-induced atheroprotection in LDLrKO mice was independent of leukocyte
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GPR120 expression by several measurements, including aortic root intimal area and whole
aorta cholesterol content.
Future Studies
PUFA-induced attenuation of inflammation involves multiple pathways. Based on our
results and other animal studies, attenuation of macrophage/Kupffer cell inflammation,
monocytosis, and neutrophilia via activation of GPR120 may have implications for improving
chronic inflammation associated with hight fat diet feeding 34, 47. Although lipopolysaccharide
(LPS) is known to activate Toll-like receptor 4 (TLR4), during chronic inflammation, aseptic
activation of TLR4 by saturated fatty acids can also activate the canonical NF-κB
proinflammatory pathway (Figure 1, pathway 1). GPR120 activation by n-3 and n-6 PUFAs
attenuates NF-κB pathway due to GPR120 internalization via binding with β-arrestin2. The
internalized complex then binds TAB1 (TAK1 binding protein 1), effectively blocking TAB1
binding to TAK1 (transforming growth factor-β activated kinase) required for activation of the
NF-κB pathway (Figure 1, pathway 2). Recent evidence suggests that inactivation of NLRP3
inflammasome (a large multimeric protein complex that is activated by pathogen-associated
molecular patterns) occurs due to GPR120 (Figure 1, pathway 2) activation and may mediate
part of the anti-inflamatory effects of PUFAs39. Whether n-3 and n-6 PUFAs equally attenuate
inflammasome activation is unknown. Additionally, since GPR120 is highly expressed in
adipose tissue and macrophages, whether adipose tissue macrophage inflammasome
inactivation by PUFAs protects from obesity and insulin resistance needs to be determined.
Autophagy is a homeostatic process that degrades cytosolic macromolecules,
damaged organelles and pathogens, whereas lipophagy is a lysosomal degradative pathway for
TG and cholesteryl esters in lipid droplets. Both autophagy and lipophagy can regulate
intracellular lipid trafficking and storage (reviewed in 48, 49). Inhibition of autophagy results in
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increased intracellular lipid storage in the form of lipid droplets and attenuation of fatty acid β-
oxidation50, whereas n-3 PUFA can induce activation of autophagy via AMPK pathway51 (Figure
1, pathway 3). Hence, autophagy likely mediates part of the lipid lowering effects of n-3 PUFAs.
Activation of autophagy can also limit inflammasome activation by degrading the inflammasome
and pro-interleukin 1-β (IL-1β) (Figure 1, pathway 4) 52, 53. Secondly, since n-3 PUFAs
inactivate NLRP3 inflammasome, activation of autophagy may be a pathway by which n-3
PUFAs regulate both inflammation and lipid metabolism and this pathway needs to be further
studied in context of n-3 and n-6 PUFAs.
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Figure 1.
.
Figure 1. Summary of potential macrophage anti-inflammatory pathways for botanical
PUFAs. (1) TLR4 activation by LPS or saturated fatty acids such as palmitate activates
canonical NFκB pathway via interaction of TAB1 and Tak1 adapter proteins, resulting in
increased pro-inflammatory gene transcription. (2) GPR120 activation by n-3 and n-6 PUFA
results in internalization via in β-arrestin2. The GPR120-β arrestin complex binds TAB1, and
inhibits NFκB activation by preventing TAB1 -TAK1 interaction. A similar pathway inhibits
NLRP3 inflammasome activation with β arrestin binding to NLRP3, resulting in decreased
processing of pro-caspase 1 and pro-IL-1β, and decreased secretion of mature IL-1β. 3) n-3
and n-6 PUFAs or their oxidized derivatives, increase AMPK activation resulting in activation of
autophagy. LPS binding to TLR4 also activates autophagy which is inhibited by palmitate via
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AMPK. 4) Activation of autophagy can inhibit inflammation by inducing NLRP3 inflammasome
degradation and pro-IL-1β degradation. Red arrows denote proinflammatory pathways; green
lines denote various pathways activated by PUFAs, black lines denote the pro-inflammatory
pathways blocked by PUFAs and blue lines denote potential pathway by which PUFA-induced
autophagy may inhibit inflammation.
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CURRICULUM VITAE
SWAPNIL VIJAY SHEWALE
230 Ridge Forest Ct, Winston-Salem, NC 27104 978-729-0185
[email protected]
HTTPS://WWW.LINKEDIN.COM/PUB/SWAPNIL-SHEWALE/19/B5B/938
Education
2007 Bachelor of Pharmacy/ Pharmaceutical Sciences, Pune University, India
2010 Master of Sciences, Boonshoft School of Medicine, Dayton, OH
2015 PhD, Wake Forest University, Winston Salem, NC
Honors and Awards
2008-2010 Fellowship: Graduate Research Assistant, Department of Pharmacology
and Toxicology, Boonshaft School of Medicine, Dayton, OH
2010 Graduate Student Excellence Award, Wright State University, Dayton, OH
2011 Best Poster, Integrative Category, Annual Research Retreat,
Wake Forest University
2012 Treasurer and Dept. Representative,
Graduate Student’s Association,Wake Forest University
2013-2014 Chair, Graduate Student’s Association, Wake Forest University
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2013 Member: Wake Forest Team, 4th Annual Biotech Case Competition
(runner up team)
2014 Team Leader: Wake Forest Team, 5th Annual Biotech Case Competition
Positions and Titles
2005 Undergraduate Research Fellow, VP School of Biotechnology, Pune,
India
2006 Undergraduate Research Fellow, National Chemical Laboratory, Pune,
India
2007 Summer Pharmacy Intern, Blue Cross laboratories, Nashik, India
2007 Key Accounts Manager, Baxter Transfusion Therapeutics, Delhi, India
2008 Project Manager, Criterium Clinical Research, Pune, India
2013-2015 Senior Commercialization Intern, Wake Forest Innovations
2011-2015 Teaching Intern, Physical Therapy program, Winston Salem State
University, NC
Travel Awards
2012 Travel Award, Annual Botanical Centers Meeting, NIH, Bethesda, MD
2014 Travel Award: Gordon Research Seminars: Lipoprotein Metabolism, NH
2014 Travel Award: Kern Lipid Conference, CO
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Oral Talks
2013 South East Lipid Research Conference, Pine Mountain, GA
2014 MHI/IVB 2014 Symposium, UNC McAllister Heart Institute, NC
2014 Columbia University, Department of Molecular Medicine, NY
2014 Lipoprotein Metabolism Section, National Heart, Lung and Blood Institute,
NIH, DC
2014 Perelman School of Medicine, University of Pennsylvania, PA
Professional Societies
2009-Current Member, American Heart Association
2013-Current Member, American Society for Pharmacology and Experimental
Therapeutics (ASPET)
2013-Current Member, American Physiological Society (APS)
2015 Member, Association of University Technology Transfer Managers (AUTM)
Publications
1. Swapnil V. Shewale, Elena Boudyguina, Xuewei Zhu, Lulu Shen, Patrick M. Hutchins,
Robert M. Barkley, Robert C. Murphy, John S. Parks. Botanical oils enriched in n-6 and n-
3 fatty acid products of FADS2 are equally effective in preventing atherosclerosis and
hepatosteatosis in mice. J Lipid Res. 2015 Apr 28. pii: jlr.M059170
Page 200
188
2. Xin Bi, Xuewei Zhu, Chuan Gao, Shewale SV, Qiang Cao, Mingxia Liu, Elena
Boudyguina, Hermina Borgerink, Martha D. Wilson, Amanda L. Brown, and John S. Parks.
Myeloid Cell-Specific ABCA1 Deletion Has Minimal Impact on Atherogenesis in
Atherogenic Diet-Fed LDL Receptor Knockout Mice. Arterioscler Thromb Vasc Biol.
2014;34:1888-1899
3. Brown AL, Zhu X, Rong S, Shewale S, Seo J, Boudyguina E, Gebre AK, Alexander-Miller
MA, Parks JS. Omega-3 fatty acids ameliorate atherosclerosis by favorably altering
monocyte subsets and limiting monocyte recruitment to aortic lesions. Arterioscler Thromb
Vasc Biol. 2012 Sep; 32(9):2122-30.
4. Shewale S, Anstadt MP, Horenziak M, Izu B, Morgan EE, Lucot JB, Morris M. Sarin
causes autonomic imbalance and cardiomyopathy: an important issue for military and
civilian health.
J Cardiovasc Pharmacol. 2012 Jul; 60(1):76-87.
5. Senador D, Shewale S, Irigoyen MC, Elased KM, Morris M. Effects of restricted fructose
access on body weight and blood pressure circadian rhythms. Exp Diabetes Res. 2012;
2012:459087.
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Published First Author Abstracts
6. Swapnil V Shewale; Elena Boudyguina; Xin Bi; Xuewei Zhu; Nilamadhab Mishra; Da Young
Oh; Jerrold Olefsky; John S. Parks. Role of Leukocyte GPR120 in Atherosclerosis
Development in Polyunsaturated Fatty Acid Diet Induced Atheroprotection in LDLrKO Mice.
Arterioscler Thromb Vasc Biol. 33: A121, 2013
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