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2008
Recommended Citation
Davis, Jeremy E., "Development of obesity-induced inflammation and insulin resistance: The role of adipose tissue, fatty acids, and toll-
like receptors" (2008). Graduate Theses and Dissertations. Paper 11169.
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Development of obesity-induced inflammation and insulin resistance: The role of
adipose tissue, fatty acids, and toll-like receptors
by
Jeremy E. Davis
DOCTOR OF PHILOSOPHY
Ames, Iowa
2008
TABLE OF CONTENTS
LIST OF FIGURES iv
LIST OF TABLES v
ABSTRACT vi
Introduction 110
Materials and Methods 113
Results 116
Discussion 119
References 123
LIST OF FIGURES
Figure 1. Fatty acid signaling through Tlr2 and Tlr4 in adipocytes. 4
Figure 2. Development of Metabolic Syndrome (MS). 10
Figure 2. Tlr4 deficiency protects mice from increased adiposity due to consumption of a
diet high in saturated fat. 105
Figure 7. Tlr4 deficiency improves markers of insulin sensitivity and inflammation in mice
fed a diet high in saturated fat. 106
Figure 4. Accumulation of F4/80-positive macrophages is attenuated in adipose tissue of
high Tlr4 deficient (10ScN) mice fed the HFP diet. 107
Figure 5. Adipose tissue inflammatory gene expression in Tlr4 deficient (10ScN) mice is
differentially regulated in response to the HFP diet. 108
Figure 6. Tlr4 deficiency attenuates NFB activation in adipose tissue of Tlr4 deficient
(10ScN) mice. 109
Figure 7. Palmitate, ZymA, and LPS induced insulin resistance in 3T3-L1 adipocytes. 130
Figure 8. Palmitate, ZymA, and LPS induced JNK phosphorylation and NFB and AP-1
nuclear translocation in 3T3-L1 adipocytes. 131
Figure 9. JNK inhibitor, SP600125, attenuated palmitate, ZymA, and LPS induced JNK
phosphorylation and inflammation in 3T3-L1 adipocytes. 132
Figure 10. JNK inhibitor, SP600125, attenuated palmitate, ZymA, and LPS induced NFB
and AP-1 nuclear translocation, oxidative stress, and insulin resistance in 3T3-L1
adipocytes. 133
Figure 11. Tlr2-/- and 10ScN mice are protected against DIO. 154
Figure 12. Absence of Tlr2 and Tlr4 expression differentially modifies markers of systemic
insulin sensitivity and inflammation. 155
Figure 13. Absence of Tlr2 and Tlr4 expression protects against SFA-induced inflammation
in AT. 156
-/-
Figure 14. SV cells isolated from Tlr2 exhibit improved insulin sensitivity, but are not fully
protected against FA-induced insulin resistance. 157
v
LIST OF TABLES
Table 1. Diet Composition 102
Table 2. Primer Sequences for Quantitative Real-Time Polymerase Chain Reaction 103
Table 3. Adipose tissue fatty acid profiles in normal and Tlr4 mutant mice fed low fat
control, high fat control, and high fat-palmitate diets for 16 weeks 104
Table 4. Palmitate, ZymA, and LPS induced inflammation in 3T3-L1 adipocytes 128
Table 5. Palmitate, ZymA, and LPS induced inflammation in 3T3-L1 adipocytes 129
Table 6. Diet Composition 150
Table 7. Primer sequences for quantitative real-time polymerase chain reaction 151
Table 8. Lipid profiles in Tlr2 and Tlr4 deficient mice and wild type controls fed LFC, HFC,
or HFP diet for 16 weeks 152
Table 9. Serum fatty acid profiles in Tlr2 and Tlr4 deficient mice and wild type controls fed
LFC, HFC, and HFP diet for 16 weeks 153
vi
ABSTRACT
The innate immune receptors, toll-like receptor-2 (Tlr2) and toll-like receptor-4
(Tlr4), are implicated in the development of inflammation and insulin resistance in various
cell populations. However, little is known about the relationship of these receptors in diet-
induced obesity (DIO). We have shown that Tlr4 deficient C57BL/10ScN mice were
protected against DIO, specifically when mice were fed a high saturated fatty acid (SFA)
diet. Moreover, these mice exhibited a specific reduction in adipose tissue (AT)
proinflammatory gene expression, and macrophage accumulation. There was also a marked
increase in Tlr2 expression in mice fed a high fat diet. 3T3-L1 adipocytes treated with the
Tlr2 agonist, zymosan A (ZymA), exhibited increased NFB and activator protein-1 (AP-1)
and insulin resistance, similarly to palmitate or Tlr4 agonist lipopolysaccharide (LPS). These
effects were attenuated in adipocytes treated with a specific c-jun N-terminal kinase (JNK)
inhibitor (SP600125) indicating involvement of this intracellular kinase in SFA and Tlr
agonist-mediated signaling. Finally, we demonstrated that Tlr2-/- mice were also protected
from DIO. However, unlike Tlr4 deficient 10ScN mice there were no significant changes in
body weight, energy intake, or lipid profile. Inflammatory and oxidative stress markers in
1)) and AT (Tumor necrosis factor (TNF), interleukin-6 (IL-6), MCP-1, F4/80, and nitric
oxide synthase-2 (NOS2) transcript abundance) were attenuated in Tlr2-/- and 10ScN mice,
which was more apparent in mice fed high SFA diet. Insulin sensitivity was improved in
10ScN, but not Tlr2-/- mice. However, stromal vascular (SV) cells isolated from AT of Tlr2-/-
vii
mice had a marked increase in insulin sensitivity, demonstrating the potential involvement of
Tlr2 in obesity-induced insulin resistance in AT. Based on these studies, Tlr2 and Tlr4 may
attenuate obesity and related co-morbidities, including Type 2 diabetes mellitus (DM) and
Introduction
Currently, approximately 60 million adults in the U.S. are classified as obese (BMI > 30),
and equally alarming, the prevalence of obesity has increased 30% during the past decade
(1). The medical and economic implications of obesity are exacerbated by its co-morbidities,
which include diabetes mellitus (DM) and atherosclerotic cardiovascular disease (CVD).
Generally, a chronic inflammatory state develops in association with obesity, and likely
contributes to the development of insulin resistance and its progression to DM (2,3). This
interleukin-6 (IL-6) (4,5). The exact mechanisms which regulate this inflammation are not
yet clearly defined, but recent evidence has unequivocally implicated the adipocyte and its
Several genetic and diet-induced rodent models of obesity have revealed that obesity
and that this may be mediated by activation of nuclear factor kappa B (NFB) or activator
hypertrophy has also been associated with increased cell death and recruitment of
subjects (14).
inflammation in AT. Toll-like receptors (Tlrs) represent key innate immune receptors that are
and Tlr4 expression are increased with obesity and DM (16). Collectively, recent findings
IL-6, and MCP-1 in adipocytes and preadipocytes through a NFB dependent pathway
(15,18,19). LPS also increased Tlr2 transcript abundance in adipocytes (20). The interaction
between the LPS molecule and Tlr4 is mediated through the lipid A component of LPS,
which consists of several fatty acids attached to a phosphate and a central glucosamine dimer
(21). The fact that fatty acids can directly interact with Tlr receptors and stimulate
downstream signaling pathways that lead to inflammation is therefore not surprising (Figure
1). Palmitate and other saturated fatty acids (SFA) clearly stimulate proinflammatory
inflammation to fatty acid signaling through Tlr4. Shi and colleagues (25) have shown that
Tlr4-/- mice have improved insulin sensitivity and attenuated inflammatory markers in AT
when stimulated with either lipid infusion or a high fat diet (25). These data are further
responses in adipocytes are attenuated when Tlr4 signaling is blunted by siRNA strategies.
More recently, Lee et al. (26) observed that SFA, but not polyunsaturated fatty acids
3
(PUFA), activated Tlr2 dimerization and activatied stress-linked kinases, such as NFB and
AP-1, in RAW264.7 macrophages. Thomas and colleagues (27) also demonstrated that the
Tlr2 ligand, Zymosan-A (ZymA), when administered at low doses in vivo, stimulated chronic
low grade inflammation contributing to the formation of new adipocytes. These studies
underscore the important link between obesity and innate immunity, and how dietary SFA
may exacerbate obesity and its related co-morbidities through direct interactions with Tlr2
and Tlr4 in AT. The significance of the current studies is that it will identify the involment of
Tlr2 and Tlr4 in obesity-induced inflammation. Moreover, elucidation of ATs role in Tlr-
Our goal is to elucidate the specific mechanisms by which SFA induce insulin
resistance in adipocytes in relation to obesity and diabetes. Our central hypothesis is that
with not only Tlr4, but also through Tlr2, and that the SFA signaling through Tlr2 also
culminates in activation of proinflammatory transcription factors NFB and AP-1 in AT. The
rationale for the work proposed herein is that understanding the relationship between innate
immunity and obesity will allow recommendations for specific prevention and intervention
strategies, and future recommendations directed at optimizing human nutrition and health
and thereby reducing the risk of diseases associated with obesity. The Specific Aims are: (i)
to determine whether Tlr2-/- and/or Tlr4-/- mice are protected against high fat diet-induced
obesity, inflammation, and insulin resistance, (ii) to determine involvement of Tlr2 and/or
4
Tlr4 signaling in adipocyte inflammation and insulin resistance, and (iii) to determine the
ability of specific dietary fatty acids to induce inflammation through Tlr2 and/or Tlr4.
Figure 1. Fatty acid signaling through Tlr2 and Tlr4 in adipocytes. Our proposed
mechanism of action of Tlr2 and Tlr4 mediated inflammation in adipocytes. Activation of
Tlrs requires stimulation by respective ligands, which include SFA. Following ligand binding
an inflammatory signaling cascade is initiated resulting in activation of proinflammatory
transcription factors, NFB and AP-1. The subsequent increase in transcription leads to the
release of proinflammatory mediators, which can act in an autocrine (e.g. adipocyte),
paracrine (e.g. adipocytes, macrophages, endothelial cells, and other AT-based cells), and
endocrine (e.g. muscle and liver tissue) manner.
5
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420: 333-336.
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12. Kanda, H., Tateya, S., Tamori, Y., Kotani, K., Hiasa, K., Kitazawa, R., Kitazawa, S.,
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Attenuation of obesity-induced adipose tissue inflammation in C3H/HeJ mice carrying a
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7
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Thompson, E. W. & Morrison, W. A. (2007) Zymosan-induced inflammation stimulates neo-
adipogenesis. Int J Obes (Lond).
8
Over the past few decades the prevalence of obesity has risen dramatically
worldwide. According to the Center for Disease Control, more than 20 states had obesity
prevalence rates greater than 25% in 2006. Data from the 2003-2004 National Health and
adolescents and adults were 17.1% and 32.1%, respectively (28). These data indicate a 3%
and gender with interactions among these factors. For instance, African Americans and
Mexican Americans show greater obesity prevalence with reduced SES, whereas this
relationship is not evident in Asian Americans (29). Regardless of these distinctions among
ethnic groups, there appears to be a significant rise in obesity independent of SES. Moreover,
the trend is expected to continue with obesity prevalence possibly reaching 35%, 36%, 33%,
and 55% by 2010 among white men, white women, black men, and black women,
respectively (30).
Obesity is a major health concern in the U.S., as well as in the majority of developed
countries. Individuals with a high BMI (>35) are estimated to have a significant reduction in
life span, especially in younger adults (31). In white males, this reduction in life span
approaches 22%. The increased mortality rates observed with obesity may be due to its
Severely obese adults (BMI >40) exhibit 7.38 times greater risk of developing DM, 6.38
times greater risk of developing HTN, and 1.88 times greater risk of developing dyslipidemia
as compared to healthy weight adults (BMI 18.5-24.9) (32). Furthermore, the prevalence
rates of DM have closely paralleled the increase in obesity. According to the CDC, the
number of individuals diagnosed with DM in the U.S. has increased from 5.6 million in 1980
to 15.8 million in 2005 independent of age, gender, and ethnicity. The majority of this
The impact of obesity and related co-morbidities on society includes not only the
health consequences, but the financial burden. From 1987 to 2001, the health care costs in
the U.S. have risen dramatically. Obese individuals account for 27% of this inflation,
medical costs approached 93 billion dollars, half of which was paid through Medicaid and
Medicare (34). The financial burden of obesity and DM also affects private companies and
it accounts for 21% of total medical costs primarily through increased disability and
absenteeism (35). Of all obesity related co-morbidities, DM represents the largest burden to
the health care system. The American Diabetes Association (ADA) reported that in 2002, the
direct (medical care and services) and indirect (disability and premature death) costs were
over 132 billion dollars. However, it has been demonstrated that these costs can be greatly
reduced with the advent of prevention vs. treatment programs targeted at addressing obesity
Referred to as the metabolic syndrome (MS), this cluster of abnormalities involves several
adiposity (Figure 2). The Third Report of the National Cholesterol Education Program
Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults
(ATP III) defines MS as three or more of the following criteria: abdominal obesity (waist
11
low high-density lipoprotein (HDL) cholesterol (1.04 and 1.29 mmol/L in men and women,
respectively); HTN (systolic blood pressure >130 and diastolic blood pressure >85 mm/Hg);
impaired glucose tolerance (IGT) (6.1 mmol/L). Based on these criteria, estimate indicate
that > 20% of U.S. adults have MS, which translates to more than 47 million Americans (37).
Substantial evidence now indicates that insulin resistance may be the primary defect
contributing to the development of IGT, dyslipidemia, and HTN. Previous investigations (38)
indicated that prevalence rates for insulin resistance in subjects with IGT,
intolerance, dyslipidemia, and hypertension was 95.2% (38). Insulin resistance, defined as
reduced sensitivity to the action of insulin, is associated with impaired glucose uptake in
peripheral tissues, particularly in skeletal muscle (39,40). The condition is also frequently
(41,42). Older, insulin resistant subjects exhibit greater accumulation of AT in the abdominal
insulin sensitivity (41). Additionally, obese postmenopausal women (body fat >45%)
exhibiting preserved insulin sensitivity have nearly 50% lower visceral AT (VAT) compared
to gender, age, and body fat-matched insulin resistant subjects (43). Accumulation of VAT is
12
also associated with the development of MS in normal weight, overweight, and obese elderly
men and women (44). The role of subcutaneous AT (SAT) in the pathogenesis of insulin
resistance and MS is less pronounced than that of VAT (45). In a review of 23 intervention
strategies (46), VAT loss in response to diet, drug, and exercise intervention represented the
highest percentage of weight loss than all other AT depots. This may be due to various
genetic and physiological differences between depots. Vohl et al (47), reported that in
severely obese subjects (BMI 45-81) nearly 350 genes were differentially regulated between
VAT and SAT. Furthermore, VAT has been shown to be more lipolytic and catecholamine
sensitive, as well as more insulin resistant, than SAT (48). These studies indicate that in
related co-morbidities, including DM and CVD (49-56). Additionally, several studies have
significantly higher in Japanese men and women diagnosed with MS than their non-affected
counterparts. In this population, the odds ratio for having MS was also greatest in individuals
categorized in the highest tertile of CRP (52). Data from the Insulin Resistance
indicated by increased CRP, is associated with dyslipidemia, central adiposity, HTN, and
insulin resistance. These findings are consistent in African-American, Hispanic, and White
13
Non-Hispanic populations. Increased CRP levels are also associated with higher age-
adjusted risks for developing DM, even after adjusting for presence of CVD, SES, physical
activity, alcohol consumption, smoking, and family history of DM (54). In subjects with DM,
leukocytes, fibrinogen, and CRP (55). Inflammation is also associated in the pathogenesis of
CVD (50,51,57). Results from the Cardiovascular Health Study (57) indicated that in adults
(>65) with atherosclerosis in combination with increased CRP had a 72% greater risk for
CVD-related death. This risk can be exacerbated in subjects diagnosed with DM, suggesting
an even greater incidence of CVD-related mortality in patients with both inflammation and
DM (58).
A novel approach for the treatment of DM and CVD is the use of anti-inflammatory
been used for their ability to reduce blood glucose (59-61). The exact mechanism is not fully
understood, but may be related to reduced FFA concentrations (59,62). One early
glucose challenge. This improvement in glycemic control was associated with a greater
insulin response and reduced FFA (63). More recently, data from the Physicians' Health
myocardial infarction with aspirin treatment compared to placebo (64). The development of
atherosclerotic CVD and stroke is increased in individuals with DM. However, the ability of
NSAIDs to reduce CVD- related morbidity and mortality in diabetic individuals remains
unclear (65). Data from the DECLARE-DIABETES Trial (66) indicated that aspirin in
14
combination with two other anti-inflammatory medications was able to lower cardiovascular
related complications in diabetic patients. Hundal and colleagues (67,68) observed that two
weeks of aspirin treatment improved insulin sensitivity and the lipid profile in male and
female diabetics. This improvement may be mediated in part by reduced IB kinase (IKK)
activation (68), which is the upstream activator of the proinflammatory transcription factor,
NFB. Activation of IKK, either in obese mice or IKK agonist treated Fao hepatoma cells,
resulted in reduced insulin signaling which was reversed by NSAID treatment (68). Overall,
including insulin resistance and dyslipidemia, which are major risk factors for CVD and DM.
IL-6. Proinflammatory cytokines, such as IL-6 and TNF, are small proinflammatory
proteins that are increased with obesity (4,5,69). The importance of IL-6 in obesity-induced
the IL-6 promoter (70-73). Fernandez-Real et al. (71) identified a SNP in the 5 flanking
region of the IL-6 gene at position -174 in which a guanine was replaced by a cytosine. This
SNP was associated with reduced plasma IL-6, as well as lower circulating insulin, TAG,
and FFA in healthy subjects. This SNP was further associated with greater insulin sensitivity
and glycemic control, as well as a reduced WBC (70). Alternatively, Kubaszek and
colleagues (72), found this IL-6 SNP to be associated with reduced energy expenditure (EE),
basal metabolic rate (BMR), whole body glucose uptake, and glucose oxidation in a healthy
Finnish population (72). These findings were further supported by a recent study (73) that
indicated this SNP is associated with higher risk of developing obesity-related metabolic
circulating IL-6 is associated with obesity related co-morbidities. Data from the Health,
Aging and Body Composition Study (74) indicated the odds ratio of developing subclinical
CVD was greatest in the tertile of patients with the highest concentration of serum IL-6.
Additionally, Bahrami, et al (75) suggested that serum IL-6 was the strongest predictor of
obesity-associated coronary heart disease. Recent data from the Monitoring of Trends and
Augsburg (KORA) study also indicated that both high IL-6 and CRP concentrations are
associated with increased mortality in elderly adults (77). The impact of IL-6 on obesity-
al (78) observed that IL-6 concentrations is positively associated with reduced -cell function
insulin signaling (80-83). Chronic IL-6 administration has been shown to inhibit insulin
signaling in adipocytes, muscle, and liver (82-84). Specifically, IL-6 was observed to inhibit
autophosphorylation of the insulin receptor (IR) and downstream effectors including insulin
(Akt/PKB), and glucose transporter-4 (GLUT4) (80-84). These effects were mediated
of cytokine signaling-3 (SOCS3) (81,82,84) and protein kinase C (PKC) (82). Activation of
PKC activation increased serine phosphorylation of IRS-1/2 (82) and thus contributed to the
development of IL-6-mediated insulin resistance. These data suggest that IL-6 may induce
signaling.
specific SNPs in the TNF gene have been associated with DM (87-89). Moreover, certain
Bisson et al. (89) identified a SNP in the TNF promoter (-238G>A) that is associated with
reduced FFA clearance and insulin resistance in obese subjects with DM but not in lean
controls. Additionally, the same SNP was observed at a higher frequency in patients with
coronary artery disease, especially in subjects with DM or HTN. Analysis of the TNF gene
and promoter indicates the importance of this cytokine in the regulation of obesity induced
insulin resistance.
Elevated TNF levels are closely related to components of MS including, IGT, HTN,
and dyslipidemia in obese adults (90) and adolescents (91). High serum TNF levels are also
associated with insulin resistance in elderly patients (92). Tam and colleagues (93) observed
that neutralization of TNF with NSAIDs contributed to improved lipid profile and insulin
as well as decreased IRS-1/2 activation (94-96). The mechanism for reduced tyrosine
extracellular signal-regulated kinases (ERK) (99), c-Jun N-terminal kinases (JNK) (100), and
p38-MAPK (97). However, the exact pathway involved appears to be tissue-specific. These
data implicate TNF as a major contributor to insulin resistance and further supports the idea
MCP-1. Chemokines are small proteins similar to cytokines, which also exhibit the
ability to induce chemotaxis. Certain chemokines, such as MCP-1 and interleukin-8 (IL-8),
promote migration of monocytes and neutrophils, respectively. Data from the Cooperative
was associated with increased risk of CVD mortality in a 7-year longitudinal study (102).
Recent work (103) has shown that a high concentration of circulating MCP-1 was associated
with insulin resistance and DM. Several SNPs have been identified in the MCP-1 gene and
promoter. Although there is still debate regarding the extent of the association, there is
evidence to suggest that the A-2518G polymorphism in the distal regulatory region of MCP-
1 is associated with heart disease and myocardial infarction (104,105). In addition, the G/G
genotype at position -2518 in the MCP-1 promoter was associated with lower plasma MCP-1
this chemokine or its corresponding receptor (CCR2) stimulated insulin resistance and
18
hepatic steatosis in several rodent models of obesity (12,106). Furthermore, multiple obese
mouse models, either transgenic (TG) or diet-induced (DIO), exhibited elevated MCP-1,
which was associated with greater macrophage accumulation into peripheral tissues,
including AT (12,106). More recently, it was observed that in vivo infusion of chemokine
stimulated adipogenesis in mice (107). This effect may account for the increased adiposity
There is limited evidence implicating the direct involvement of MCP-1 with insulin
glucose homeostasis, including the pancreas, liver, skeletal muscle, or AT, results in an
Additionally, MCP-1 involvement in oxidative stress and ROS formation represents another
1 expression in pancreatic islets and beta-cells could result in -cell dysfunction and thus
promote IGT. Regardless of the mechanism, it is clear that increased MCP-1 expression is
ADN. Obesity is not only associated with increased proinflammatory cytokines and
proteins, such as adiponectin (ADN). Several genetic analyses (109,110) observed that a
quantitative trait locus (QTL) on chromosome 3 (3q27), the site of the ADN gene, was
strongly correlated with insulin resistance, MS, and DM. Data from the Metabolic Risk
Complications of Obesity Genes Project (109) indicated that chromosome 3 also had the
highest association with BMI, waist circumference, and insulin sensitivity. Additional
genetic analysis observed the presence of multiple SNP in the ADN gene associated with
19
increased risk of DM and CVD (111-115). More specifically, the G/G genotype at position
276 in the ADN gene is associated with reduced ADN, as well as insulin resistance, DM, and
CVD (111-113). Multiple investigations have since indicated that obesity is associated with
dysregulation of ADN (116-118). Arita et al. (116) initially reported a negative correlation
associated with several components of MS, including insulin resistance, dyslipidemia, and
individuals with MS had lower serum ADN compared to obese individuals without MS.
Patients diagnosed with DM or CVD are also observed to have significantly lower plasma
ADN (118,121,122). This is especially true for high risk populations, such as Pima Indians.
varied metabolic effects. Plasma ADN was negatively correlated with CRP, IL-6, and TNF
in obese subjects (123,124). Furthermore, ADN attenuated LPS-induced IL-6 and TNF
activation was also prevented with ADN treatment (22), which may explain the observed
monocyte proliferation and macrophage function, including phagocytic activity and cytokine
release, were reduced by ADN (130,131). Foam cell formation, as determined by lower
macrophage cholesteryl ester content and scavenger receptor expression, was also attenuated
by ADN (132). These data indicate that ADN is a key anti-inflammatory mediator.
Several studies have examined the role ADN plays in vivo. Genetically engineered
ADN knockout (KO) mice exhibited reduced insulin sensitivity in response to a high fat diet
20
(133-135). Maeda et al. (133) demonstrated that ADN deficient mice had impaired insulin
signaling in skeletal muscle. Moreover, this effect was corrected by adenoviral produced
ADN (133). Alternatively, Nawroki and colleagues (134) suggested that the reduction in
insulin sensitivity in ADN KO mice was attributed to increased hepatic glucose production.
Several additional studies (136,137) indicated the liver as the primary site of ADN action.
structural differences in the protein. ADN forms several distinct oligomeric complexes.
These include a high molecular weight (HMW, >250 kDa), medium molecular weight
(MMW or hexamer, ~180 kDa), and low molecular weight (LMW or trimer, ~90 kDa)
species (138-141). Furthermore, a proteolytic cleavage product of ADN that includes the
globular C-terminal of the protein (gADN) was also present in human plasma (142).
In humans, females have a greater proportion of HMW and hexamer complexes than
males (138-140). Additionally, BMI is negatively correlated with the concentration of HMW
species, but not the hexamer or trimer forms (139). Pajvani and colleagues (138) reported
that lower HMW to total ADN was associated with lower insulin sensitivity in diabetic
increased HMW but not total ADN in these subjects (138). The HMW species is suggested to
improve hepatic insulin sensitivity (138,141). Wang et al. (141) demonstrated the HMW
species stimulated 5'AMP-activated protein kinase (AMPK) in liver, which lead to inhibition
phosphatase (G6Pase). Alternatively, gADN was associated with skeletal muscle insulin
sensitivity in obese and diabetic humans (143,144) and rodents (142,145-147). Bruce and
colleagues (144) observed that gADN in vitro stimulated glucose uptake and FA oxidation in
21
skeletal biopsies from lean and obese subjects. This effect was attributed to increased AMPK
and IRS-1 activation, as well as glucose uptake in primary rat adipocytes (148).
ROS are small, highly reactive molecules possessing oxidative abilities primarily
peroxide (H2O2), hydroxyl radical (OH), and nitric oxide (NO) (149). These molecules play
important roles in cell signaling. However, accumulation of ROS can contribute to oxidation
of amino acid side chains, protein-protein cross-links, and protein fragmentation (149).
investigations have indicated a role of oxidative damage in atherosclerosis (151), HTN (152),
and DM (153). Obesity has also been linked to increased oxidative stress (154,155). Data
from the Framingham Heart Study (155) indicated that urinary 8-epi-prostaglandin F2 alpha
pathway, was positively associated with BMI and waist circumference in male and female
subjects. Moreover, for every 5 kg/m2 of BMI there was a 9.9% increase in urinary 8-epi-
PGF2 concentration, indicating very high levels of oxidative stress. Furukawa et al. (154)
also observed a positive correlation between BMI and urinary 8-epi-PGF2 in healthy
Japanese men and women. Additionally, a positive association has been shown (154)
between obesity and plasma thiobarbiturate reactive substances (TBARS), an end product of
lipid peroxidation.
Obesity-induced oxidative stress represents a potential link between obesity and its
associated co-morbidities. Cardona and colleagues (156) reported that individuals with MS
22
exhibited increased markers of oxidative stress compared to patients without MS. This same
group also demonstrated that antioxidant defense systems were impaired in healthy
individuals following high fat feeding (157). The increased oxidative stress associated with
individuals diagnosed with DM exhibit increased oxidative stress and reduced antioxidative
capacity compared to healthy controls (158). Mohora et al. (159) determined that obesity and
DM were both associated with increased ROS, but obese subjects with DM exhibited a
findings are supported by additional groups (160) and clearly link oxidative stress as a bridge
The mechanisms involved in oxidative stress-induced insulin resistance have not been
hepatocytes (161), skeletal muscle (162-164), and adipocytes (165-167). Kumashiro and
dismutase 1 (SOD1) in the liver of diabetic db/db mice reduced hepatic ROS accretion and
improved insulin sensitivity. Additionally, Dokken and colleagues (163) observed that
isolated rat skeletal muscle incubated with H2O2 significantly inhibited insulin-stimulated
to oxidative stress (165-167). Rudich et al. (165) showed that low grade oxidative stress
adipocytes. Furthermore, basal, but not insulin-stimulated, glucose uptake was increased
along with GLUT1 expression in response to increased H2O2 content (165). Several
additional studies (164,166,168) confirmed that an increase in basal glucose transport occurs
glucose uptake may exacerbate ROS accumulation and inflammatory signaling in adipocytes
(169-172) through activation of NFB and AP-1 (173). These studies clearly link adipocyte-
specific oxidative stress to the development of insulin resistance and therefore connect
humans (174-177) and animals (178,179). This elevation in FFA is largely attributed to
increased basal AT lipolysis (180-182). However, the antilipolytic effect of insulin may also
FFA is associated with insulin resistance in obese individuals (185,186). Moreover, infusion
of FFA in lean humans and rats stimulated insulin resistance (187,188). These data indicate
that an increase in FFA represents a primary connection between obesity and insulin
resistance.
parents diagnosed with DM (191), reported that non-diabetic offspring had increased FFA
24
that predisposed them to the development of DM. Pima Indians with the highest
concentration of FFA had a 2.3 fold greater risk of DM than individuals with the lowest
concentration of FFA (192). Impaired postprandial FFA clearance is also linked to CVD in
diabetic individuals (193). Carlsson and colleagues (194) observed that higher FFA were
associated with greater risk of myocardial infarction and stroke. Other markers of CVD,
including endothelial dysfunction, vascular smooth muscle cell proliferation, and HTN were
also associated with increased FFA (194). Additional studies (90,174) have indicated that
elevated FFA is associated with the development of several other components of the MS,
Insulin sensitivity can be improved by lowering FFA concentration. Studies using the
sensitivity in obese and diabetic individuals (193,195,196). Santomauro et al. (196) reported
that acipimox reduced plasma FFA by more than 60%, which improved insulin-stimulated
glucose uptake by greater than 2-fold in obese subjects under euglycemic hyperinsulinemic
(197). Data from the Bezafibrate Prevention Study showed a near 10% reduction in CVD-
related mortality (198). Moreover, it has been observed that treatment with the PPAR
agonist improved several additional complications of MS, including insulin resistance (198-
200). Haluzik and colleagues (201) demonstrated that 14 d of fenofibrate treatment reduced
blood glucose, FFA, TAG, and insulin resistance in lipogenic, simple carbohydrate diet fed
mice. However, the insulin-sensitizing response of fibrates in humans is limited, which may
25
be due to lower PPAR expression in liver and skeletal muscle (202). The ability of lipid-
lowering medication to effectively attenuate insulin resistance, as well as lower risk for
developing DM and CVD, indicates that FFA are key mediators in obesity-related co-
morbidities.
induced insulin resistance has not been fully elucidated. However, Randle et al. (203)
originally proposed that the insulin resistant properties of FA were mediated through direct
synthesis, such as citrate and acetyl-CoA, accumulate in the cytosol of muscle cells, enzymes
complex, are inhibited. This in turn reduces insulin-stimulated glucose uptake and
metabolism (204-207). Overall, researchers have suggested that increased FFA induces
develops, -cell dysfunction occurs and insulin secretion can no longer compensate for
lipid within non-adipose tissue, such as skeletal muscle, liver, and pancreatic -cells, has a
more profound effect on cellular metabolism than impairment of glucose metabolism. This is
evident by the increased level of apoptosis in these tissues in response to lipid accumulation
insulin resistance (211). The consequences of lipotoxicity may be mediated through products
been associated with the synthesis of DAG and ceramide (208,209,212). This effect appeared
to be protective (213). The free radical, nitric oxide (NO) has also been correlated with
ceramide concentration and may represent the link between the FA metabolites and -cell
apoptosis (209). In addition to the potential reduction of insulin synthesis, that DAG and
ceramide have been shown directly to inhibit insulin signaling (208,214-216). This action
may be mediated PKB and PI3K, which are key intermediates of insulin-mediated glucose
uptake (208,214,215). More specifically, FA metabolites activate PKC (208) and protein
phosphatase 2A (PP2A) (214), which are both shown to directly inhibit insulin-mediated
GLUT4 translocation. Proinflammatory cytokines, such as IL-6 and TNF, have also been
shown to increase synthesis of ceramide (208), as well as directly stimulate PKC activation
Dietary FA can be both pro- and anti-inflammatory. SFA may be responsible for the
increase in proinflammatory cytokines and chemokines observed with obesity and MS (217-
222). The consumption of a Westernized diet high in refined grains, red meat, SFA, and
hydrogenated fats has been positively related to several markers of inflammation, including
CRP, serum amyloid A (SAA), and IL-6 (223). Aeberli et al. (220) reported that total dietary
fat is associated with CRP and IL-6 in obese children. Moreover, SFA intake is specifically
27
that palmitic and myristic acid consumption were negatively correlated to plasma ADN and
improved insulin sensitivity (225-229). More specifically, Pischon and colleagues (226)
reported intake of the n-3 FAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid
(DHA), were inversely associated with plasma concentrations of the soluble TNF receptor
(sTNF-R) and CRP. Data from the Nurses Health Study (225) demonstrated that EPA and
DHA intake were also inversely related to CRP, IL-6, and several proinflammatory adhesion
factors, including E-selectin, soluble intracellular adhesion molecule (sICAM-1), and soluble
observed with consumption of a diet rich in MUFA (228,229). Fito et al. (228) observed that
consumption of virgin olive oil reduced plasma IL-6 and CRP in patients diagnosed with
coronary heart disease. Thus, substitution of dietary SFA or trans FA (TFA) with either n-3
FA or MUFA may atenuate obesity-induced inflammation and reduce the risk of DM and
CVD.
elucidated, but it is clear that certain SFAs stimulate a proinflammatory response in various
cells and tissues. Palmitate and other medium chained SFA-induced proinflammatory gene
macrophages (11), and adipocytes (22,100,235). Elevated circulating palmitate was also
positively correlated with increased plasma IL-6 (231) and negatively correlated with plasma
ADN (224). Moreover, human coronary artery endothelial cells exhibited increased IL-6
28
mRNA and protein expression in response to palmitate treatment (231). This vascular
inflammation has also been associated with development of NASH (236). Joshi-Barve et al.
(232) reported that palmitate-induced IL-8 expression stimulated lipid accumulation and
insulin resistance in cultured hepatocytes (232). These effects have been dramatically
Skeletal muscle accounts for over 80% of glucose uptake under insulin-stimulated
inflammation and insulin resistance. SFAs, such as palmitate, increased IL-6 and TNF
expression in skeletal muscle cells and have been associated with reduced insulin sensitivity
IRS-1, PI3K, and Akt/PKB phosphorylation in L6 myotubes. Furthermore, Jove et al. (240)
expression, in C2C12 muscle cells. These studies and others (233,239,241-243) indicated
activation of transcription factors, such as NFB and AP-1 (232,244,245). Blocking MAPK
resistance (233,234,239-241). Weigert and colleagues (233) reported that the proteosome
corresponding IL-6 production in human myotubes. This relationship in consistent with other
(234,240,241).
The ability of FFA to induce inflammation and insulin resistance in liver and skeletal
muscle suggests that disturbances in these tissues are dependent on interactions with
adipocytes. Dietze et al. (246) observed that co-culture of human muscle cells with
accumulation of FFA in skeletal muscle with obesity (247,248). This ecotopic storage can
then promote systemic insulin resistance (249,250). Boden and colleagues (251) suggested
that insulin resistance occured in adipocytes prior to skeletal muscle. They provided evidence
that reduced insulin sensitivity in adipocytes promoted greater lipolysis and reduced FFA
uptake that consequently led to ecotopic fat storage. Moreover, increased FFA concentration
impaired insulin signaling in adipocytes. Ajuwon et al. (22) reported that palmitate induced
IL-6 expression and NFB activation in 3T3-L1 adipocytes. FFA also increased TNF and
impact of adipocytes on inflammation and insulin resistance suggests that they represent a
AT represents one of the most dynamic tissues in the human body. It can represent as
little as 3% of total body weight in conditioned athletes to over 70% of total body weight in
severely obese individuals (252). The cellular prolife of AT is highly variable and depends
greatly on depot location, as well as multiple genetic and environmental factors. However,
despite substantial intra- and inter-individual differences there is a basic organization to AT,
nodes, and nerves (252,253). Adipocytes represent the major cellular component of AT.
There are two distinct classifications of adipocytes: the unilocular white adipocytes and
White adipocytes contain a large lipid droplet composed primarily of TAG and
cholesteryl esters, ranging in size from 20 to 200 M in diameter. Moreover, they exhibit
distinct endocrine, paracrine, and autocrine properties via expression and secretion of
multiple proteins, such as leptin, ADN, IL-6, TNF, and MCP-1. Adipocytes are also targets
for various signaling molecules released from -cells, macrophages, neurons, hepatocytes,
and adipocytes themselves. Some of the adipocyte-specific receptors mediating this signaling
include insulin, cytokine (i.e. IL-6 and TNF), adrenergic/cholinergic, and lipoprotein
receptors (254). Therefore, it is clear that the physiological role of white adipocytes extends
beyond simple energy storage and involves complex interactions with central and peripheral
Brown adipocytes have a slightly different morphology than white adipocytes. For
instance, these cells are smaller (20 to 40 M), contain multiple lipid droplets, have
31
pronounced nuclei and cytoplasm, as well as increased capillary density (255). Brown
brown color. The major functional difference between the two cell populations is the role of
brown adipocytes in thermogenesis. Briefly, FAs stored within brown adipocytes are quickly
this gradient is used for ATP synthesis, but in brown adipocytes the uniquely expressed
protonophore, uncoupling protein 1 (UCP1), mediates disruption of the gradient causing the
plays an important role in thermal regulation. Newborn infants also possess high levels of
brown adipocytes in several adipose depots (255). However, in adult humans the amount of
these cells is dramatically reduced and are found dispersed among white adipocytes. In lean
adults, researchers have suggested that, on average, there is one brown adipocyte for every
100 to 200 white adipocytes in VAT (257). Several studies (253,255) indicated that reduced
into a phenotype similar to white adipocytes. Moreover, it appears that this process may be
reversed by using -adrenergic agonists (258). The ability of these two adipocyte populations
Adipose expansion involves both adipocyte hypertrophy (increase in cell size) and
hyperplasia (increase in cell number). Hypertrophy and hyperplasia occur naturally in normal
growth phases, as well as with obesity (discussed below) (259,260). Adipocyte hypertrophy
requires positive energy balance that results in accumulation of TAG. However, adipocytes
are limited in the amount of lipid that can be stored. Hence, hyperplasia represents an
increase cell number. Alternatively, differentiation of preadipocytes requires cell cycle arrest,
partially differentiated cells may still be capable of replication (260). In normal human
the lipogenic capacity of the cell (262,263). Two transcription factors, CCAAT/enhancer
binding protein (C/EBP) and PPAR, are essential to adipocyte differentiation. Following
initiation of growth arrest, preadipocytes undergo at least one more round of DNA
replication and cell doubling, often referred to as clonal expansion (263,265). During this
Complete (or terminal) differentiation of adipocytes is associated with complete exit from
cell cycle and an increased expression of adipocyte-specific genes and proteins, including
hyperplasia (252,263,266). In obesity, mature adipocytes reach their lipid storage capacity at
adipocytes secrete growth factors in response to their expanding size (264). These growth
factors, such as insulin-like growth factor (IGF), can then promote preadipocyte
33
contributing to obesity (270). Wang and colleagues (271) observed that mice lacking
adiposity, an effect that was reversed through transgenic overexpression of the preadipocyte
treatment of obesity and related co-morbidities (272). Therefore, individuals with greater
TAG stored in AT originates from either dietary fat that is packaged into
produced TAG that is packaged into very low density lipoproteins (VLDL) in the liver (post-
absorptive) (274). Once in the circulation, these lipid rich lipoproteins deliver TAG and FA
to peripheral tissues, including skeletal muscle and AT. Delivery of FFA to adipocytes
requires hydrolysis of lipoprotein based TAG from chylomicrons and VLDL by lipoprotein
adipocytes and then exported to the luminal surface of capillary endothelium where it
hydrolyses, with acylation stimulating protein (ASP), TAG to FFA and glycerol (277).
Dysregulation of LPL, as observed in obesity has been associated with dyslipidemia and
overexpressed LPL attenuated insulin resistance in obese rabbits. However, increased LPL
activity in AT is associated with dyslipidemia and insulin resistance (279). The mechanism
34
underlying these differences is unclear, but may relate to the difference in the lipid oxidative
Following hydrolysis of TAG from lipoproteins in the endothelial lumen, FFAs are
CD36 and fatty acid transport protein (FATP). Expression of these transport proteins have
been shown to be increased in obesity and DM (280,281). Gertow and Colleagues (281)
observed that increased expression of FATP4 was positively correlated with obesity and
insulin resistance in monozygotic twins. Based on these studies, it appears that regardless of
The majority of TAG is from dietary sources; however, de novo lipogenesis (DNL)
non-lipid sources, primarily glucose. In humans, DNL occurs extensively in the liver (282)
(283). FA generated by hepatic DNL are packaged into VLDL for transport to peripheral
tissues (284). AT is also indicated as a site for DNL in humans. The contribution of DNL in
AT compared to liver is lower on a per gram of tissue basis, but considering the total amount
of AT, especially in obese individuals, the overall contribution of FA is similar between the
two tissues (285). Furthermore, in humans, high a CHO diet appears to mediate little effect
on DNL in AT (285). Alternatively, the reduction in DNL observed in liver with a high fat
diet is not seen in AT (286), indicating that DNL in AT is less susceptible to nutrition or
endocrine regulation. However, the mechanisms regulating DNL in AT and its implication in
AT is not only an important site of FA/TAG synthesis and storage but also represents
oxidized in WAT is insubstantial compared to skeletal muscle (288), the large concentration
of mitochondria found within white adipocytes indicates these cells are involved in oxidation
of FA (289). Furthermore, several studies (290) elucidated that while the amount of
to other tissues such as liver. Masseen and colleagues (287,290) suggested that FA in cytosol
of white adipocytes was cleared through mitochondrial oxidation, thus limiting release of
FFA into circulation. Moreover, they proposed that with a greater increase in cytosolic FA
circulation and thus preventing lipotoxicity (290). Mitochondrial uncoupling allows a greater
amount of FA to be removed through the generation of heat rather than ATP production. In
obesity an increase influx of FA into the cytosol of adipocytes may exceed the protective
FFA released into circulation (287,290). The subsequent increase in plasma FFA contributes
to ectopic TAG accumulation in non-AT (i.e. pancreas, liver, and skeletal muscle), which
was observed to activate AMPK signaling (294), suggesting that adipocytes regulate FA
36
in ADN and AMPK expression in adipocytes, indicating that downregulation of these genes
Lipolysis, which is the hydrolysis of TAG into FFA and glycerol in adipocytes, is
through interactions with adrenergic receptors (296). Signaling through these receptors leads
to activation of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) which
in turn activates HSL (284). The cAMP/PKA also mediates phosphorylation of the
phosphoprotein perilipin, which surrounds lipid droplets and serves as a docking protein for
HSL (284). The antilipolytic effect of insulin is mediated through increasing intracellular
characterized by high circulating FFA (185,251). This would suggest that obesity is
associated with greater levels of lipolysis. However, several studies have demonstrated that
has been shown to be dramatically reduced in obese individuals (284). This metabolic
paradox may be partially explained by an increase in basal lipolysis. The exact mechanisms
regulating basal lipolysis are not fully elucidated, but may be related to adipocyte size (299).
Therefore, adipocyte hypertrophy leads to increased basal lipolysis that in turn leads to FFA-
associated with increased TNF release from AT. This group subsequently reported that
adipose-specific TNF mRNA expression was negatively correlated with insulin sensitivity
in obese postmenopausal women (300). The source of this increased TNF expression in AT
was initially attributed to adipocytes, but several groups (6,301,302) recently reported that
the stromal vascular fraction (SVF) of AT released greater levels of TNF than neighboring
adipocytes. Cells located in the SVF include preadipocytes, endothelial cells, smooth muscle
cells, leukocytes, and macrophages. Weisberg and colleagues (7) suggested that adipose
tissue macrophages (ATM) represented the major source of TNF. Regardless of the source,
and MAPK (including ERK1/2, p38 MAPK and JNK). Activation of these pathways in
or indirect mechanisms. Several groups (78,94,95,98) have demonstrated that TNF mediates
insulin resistance in skeletal muscle. Plomgaard et al. (95) reported that infusion of TNF in
healthy humans induced insulin resistance in skeletal muscle through disruption of insulin
These data indicate the importance of this cytokine in obesity-induced insulin resistance.
However, Ofei and colleagues (305) demonstrated that TNF-neutralizing antibody failed to
improve insulin sensitivity in obese individuals diagnosed with DM. This study suggested
that in vivo circulating TNF may not be sufficient to induce obesity-induced insulin
resistance.
insulin resistance in adipocytes (306). This cytokine can block insulin-stimulated IR, IRS,
involves signaling through JNK (100) and NFB (307). This inflammatory signaling
contributed to accumulation of ROS (309), which can further exacerbate insulin resistance.
including reduced lipogenesis and increased lipolysis. The reduction in lipogenesis involves
inhibition of FA uptake are not well understood, but may involve downregulation of FATP,
as well as decreased LPL activity (310,311). The induction of lipolysis and release of FFA
stimulates lipolysis through activation of kinases such as JNK and ERK1/2, which decreased
the expression of antilipolytic genes (312). TNF-induced lipolysis may also be exacerbated
by inhibition of adipogenesis. Several groups (313,314) have reported that TNF reduced
and secrete IL-6 (22,84); however, their overall contribution to sera IL-6 concentration may
be limited as the SVF contributes a significant proportion of IL-6 released from AT (316).
Mohamed-Ali, et al. (318) reported that in obesity, adipocytes may contribute up to 30% of
total circulating IL-6. There are two major signaling pathways activated by IL-6: the Janus
Like TNF, the regulation of IL-6-induced insulin resistance in obesity may be due to
direct and/or indirect effects on peripheral tissues, including skeletal muscle and liver.
Several studies have suggested that IL-6 is responsible for hepatic insulin resistance
(81,83,237). Klover and colleagues (237) demonstrated that reductions in plasma IL-6,
mediated by neutralizing antibody specific for IL-6, were associated with improved hepatic
which include increased activation of IR, IRS, PI3K, and Akt/PKB (81,237). Moreover, IL-6
has been shown to induce STAT3 and suppressors of cytokine signaling (SOCS) (81,83,237).
(237). Interestingly, the effect of circulating IL-6 on insulin sensitivity in skeletal muscle is
less clear. In fact, minutes within starting exercise, the intracellular concentration of IL-6
increased 100-fold in muscle cells (237). Febbraio and colleagues (320) reported that this
elevation in IL-6 during exercise was associated with an increase in whole body glucose
40
uptake. Furthermore, IL-6 deficient mice show a reduced capacity for aerobic exercise (321).
Therefore, these data indicate that in terms of skeletal muscle physiology, IL-6 has an
The paradoxical relationship between circulating IL-6 and systemic insulin resistance
may be partially explained by the autocrine and paracrine effects of IL-6 on adipocytes.
Several groups (80,84) reported that IL-6 blunted insulin-stimulated IR, IRS-1, Akt/PKB,
adipocytes is dramatically impaired by IL-6 through blocking the entry of substrates (glucose
and FFA) necessary for TAG synthesis. Along with the reduction in lipogenesis is a
corresponding increase in lipolysis (323,324). Peterson and colleagues (323) observed that
acute infusion of IL-6 stimulated lipolysis in elderly subjects. The overall implication of IL-6
in adipocyte metabolism is a reduced capacity for lipid storage and increased flux of FFA
into the circulation. Thus, increased adipocyte hypertrophy stimulates IL-6 production and
release from adipocytes, which in turn exacerbates lipolysis and FFA-induced insulin
resistance in skeletal muscle. This effect is further supported by a recent study that observed
conditioned medium from mature lipolytic adipocytes that stimulate inflammation and
infiltration (3,6,7). Weisberg and colleagues (7) reported that adipocyte size was positively
correlated with expression of the macrophage marker F4/80 in AT of obese mice. In humans,
41
the macrophage marker CD68 was correlated with BMI, as well as adipocyte size (7). The
majority (85%) of adipose-specific cells are thought to be derived from bone marrow (7).
includes expression of macrophage-specific cell surface markers (i.e. F4/80 and Mac-1) and
known that these cells originate from progenitor cells in bone marrow. Furthermore, there are
two major subpopulations of ATM, which includes a short-lived subset actively recruited to
colleagues (328) also suggested that resident ATM undergo a phenotypic change in response
into AT (diapedesis) involves several steps that include: activation of capillary endothelium,
tissues (330). Within AT, macrophages localize primarily to sites of necrotic-like adipocyte
cell death and aggregate in crown-like structures (CLS) around individual adipocytes (331).
Cinti, et al. (331) demonstrated in HSL knockout mice that there was excessive adipocyte
these data, it is evident that one of the ATM functions is to clear necrotic adipocytes.
with angiogenesis (332,333). Macrophages have been shown to mediate angiogenesis during
tumor growth (334) and inflammation (335). More recently, Pang and colleagues (336)
42
adipocyte differentiation (337-339). Kim and colleagues (339) also reported that macrophage
circulating FFA (339), leading to insulin resistance in adipocytes or in peripheral tissues (i.e.,
skeletal muscle and liver). Furthermore, ATM may cause preadipocytes to exhibit a
(340).
The regulation of macrophage infiltration into AT is not fully elucidated but likely
hypertrophic adipocytes (7). Numerous chemoattractant proteins, such as MCP-1 and colony-
Leptin is also involved in endothelial cell activation and monocyte transmigration (329).
However, leptin deficient ob/ob mice still exhibited macrophage accumulation in AT (6),
43
Adipocytes, endothelial cells, and macrophages have all been observed to contribute to the
release of this chemokine from AT (69,341,342). Christiansen et al. (343) reported that
indicating that AT is the primary source of the chemokine in obesity and was associated with
insulin resistance. Westerbacka and colleagues (344) reported that obese and insulin resistant
compared to lean subjects. Collectively, these studies suggest that adipocytes release
well as increased lipolysis, thus promoting systemic inflammation and insulin resistance.
innate immune cells. In mammals, there are currently 13 identified isoforms of Tlrs (345).
Each Tlr recognizes distinct structural motifs primarily expressed on foreign pathogens such
(PAMPs) range from specific lipids to nucleic acid sequences. For instance, Tlr4 recognizes
lipid based structures, such as LPS, whereas Tlr3 recognizes double stranded viral DNA
(345,346). The cellular location of Tlrs is an important factor in determining their respective
ligands. Tlrs located on the plasma membrane (i.e. Tlr1, 2, 4, 5, 6, 10, and 11) commonly
recognize lipid or protein based PAMPs (347). Alternatively, intracellular based Tlrs (i.e.
44
Tlr3, 7, 8, and 9) most commonly recognize bacterial and viral nucleic acids (347). The wide
indicates their importance in host defense. Tlrs are expressed on immune cells involved in
innate and adaptive immunity, including macrophages, neutrophils, dendritic cells, and T and
B lymphocytes (346). More recently, expression of these innate immune receptors were
identified in cells not traditionally characterized as immune cells, such as intestinal epithelial
The activation of Tlrs through interactions with their respective ligand or PAMP
initial event following ligand binding is the recruitment of adaptor proteins to the
intracellular domain of Tlrs. These adaptor proteins, include MyD88, TIR domain-containing
adaptor protein (TIRAP), Trif, and TRAM (352). MyD88 is the most prominent adaptor
protein involved in the majority of Tlr signaling. In fact, only Tlr3 and Tlr4 are capable of
signaling independently of MyD88 (352). The more prominent MyD88 dependent pathway
involves several additional proteins including IL-1R-associated kinase (IRAK) and TNF
subsequently activates IKK and MAPK signaling pathways (347,352). Several groups
protein 1 (TAB1) leading to activation of IKK and MAPK pathways. However, the exact
Activation of IKK mediates phosphorylation of IB, marking the inhibitor protein for
also induce NFB activation through a delayed induction mediated through Tlr4 (356,357).
(352). Activation of the MAPK family, including JNK, ERK1/2, and p38, results in nuclear
translocation of the transcription factor, AP-1 (358,359). Both NFB and AP-1 are required
for cytokine production, but it is unknown how activation of these transcription factors and
distinct Tlrs.
endotoxin (or lipid A), is composed of FA and phosphate groups attached to a central
glucosamine dimer (360). A specific serum binding protein transports endotoxin to cells
expressing its corresponding receptor, which was recently identified as Tlr4 (361). The Tlr4
complex consists of the Tlr4 receptor itself and two co-receptors, MD-2 and CD14 (362).
The MD-2 co-receptor is essential to Tlr4 function (363), whereas CD14 is a high affinity
GPI-linked protein necessary for LPS binding (364). Following binding of LPS to CD14,
Tlr4 is close enough to recognize endotoxin and initiate signal transduction through MyD88-
Several studies (365-370) have examined the impact of SNP in the Tlr4 gene on the
development of CVD and DM. The findings of these studies are mixed but indicate that
46
certain polymorphisms exhibit protective effects. For instance, the Asp299Gly SNP was
associated with changes in extracellular domain of Tlr4 causing reduced sensitivity to LPS
(371). Data from the Bruneck Study (365) determined that 53 of 810 subjects were
heterozygous for Asp299Gly SNP. Furthermore, carriers of this allele exhibited lower
proinflammatory cytokines, ACP, and adhesion molecules, which was associated with lower
atherosclerotic risk (365). Edfeldt and colleagues (370) also reported that this SNP was
al. (369) reported a reduction in CVD and DM risk with individuals exhibiting the
Asp299Gly SNP. More specifically, presence of the SNP has been associated with lower
Alternatively, several studies have indicated that polymorphisms in Tlr4 have little impact on
progression of chronic disease in humans (366,367). The disparities among these studies are
CVD and DM, there is substantial evidence that the innate immune receptor is a major
mediator of obesity-induced insulin resistance. Studies using the Tlr4 ligand, LPS, clearly
showed the importance of the receptor in the development of insulin resistance in several cell
types, including myotubes (372,373), hepatocytes (374), and adipocytes (23). Frost and
colleagues (372) suggested that LPS-induced insulin resistance in C2C12 myotubes was
TNF and IL-6, respectively. Moreover, these effects were attenuated in skeletal muscle of
Tlr4 deficient mice (372). Inflammation was also attributed to the LPS/Tlr4-mediated insulin
47
resistance in hepatocytes and adipocytes (23,374). Song et al. (23) observed that LPS
stimulated TNF and IL-6 expression, which in turn resulted in impaired insulin signaling.
There is also evidence that obesity is associated with increased circulating LPS, a
major in vivo agonist of Tlr4, and that this endoxemia is a primary mediator of insulin
resistance (16,375). Cani and colleagues (375) demonstrated that plasma LPS was increased
in obese mice. In humans, serum endotoxin levels are reported to be over 75% higher in
LPS concentration was associated with insulin resistance in obesity, implicating a role of
Tlr4 in obesity-induced inflammation. For instance, Tlr4 deficient mice were protected from
(24) observed that C3H/HeJ mice, with a missense mutation in the cytoplasmic domain of
Tlr4, exhibited reduced TNF expression in AT and improved insulin sensitivity following
16 weeks of a high fat diet (HFD) (24). These findings are supported by two recent studies
(376,377). However, the impact of aberrant Tlr4 signaling on adiposity remains unclear.
Tsukumo et al. (377) reported a reduction in epidydimal fat pad weight due to increased
oxygen consumption and reduced respiratory exchange ratio, although the exact mechanisms
are unknown. Alternatively, Poggi and colleagues (376) observed an increase in adiposity in
HFD fed C3H/HeJ mice. The disparity between these studies may be due to distinct
infiltrated atherosclerotic lesions of mice and humans (380). Furthermore, Kim and
colleagues (378) observed that Tlr4 knockout mice were protected from HFD-induced
48
Overall, these studies indicate that Tlr4 may be a potential mediator of obesity-induced
Tlr2. Of the various innate immune receptors, Tlr2 recognizes the most diverse
lipoteichoic acid and peptidoglycan), polysaccharides (i.e. zymosan), and whole bacteria
(381,382). LPS was originally characterized as a Tlr2 agonist (383), but it was later
determined that this effect was mediated by contaminating lipoproteins (384). However, two
studies (385,386) reported that Tlr2 recognized atypical forms of LPS from Leptospira
interrogans and Porphyromonas gingivalis. The atypical forms of LPS differ in structure and
function than the type recognized by Tlr4 by their different number of acyl groups in the
lipid A component (387). Regardless of the exact agonist, Tlr2 activation requires
dimerization with Tlr1 or Tlr6 (388,389). The specific heterodimer formed in the Tlr2
complex appears to be dependent on the size and number of fatty acyl chains. However, post-
There are currently 16 polymorphisms identified in the Tlr2 gene. Of these, half are
non-synonymous mutations leading to amino acid exchanges and the remaining half are
(390,391) demonstrated SNPs in the Tlr2 gene that were associated with reduced cytokine
production. More specifically, Merx and Colleagues (390) reported a novel SNP in Tlr2
(arginine codon at 447 to stop codon) associated with reduced IL-6 and TNF production in
heterozygote Arg447 stop individuals. Additional genetic analyses have observed that
49
various SNP in Tlr2 were protective against several inflammatory conditions, including Type
Despite the limited genetic analysis, there is evidence to suggest involvement of Tlr2
expression of Tlr2 was increased in AT of obese diabetic individuals, whereas Tlr4 levels
were unchanged. Furthermore, expression of this innate immune receptor was associated
proinflammatory cytokine expression (16). Murakami and colleagues (394) utilized flow
cytometry to analyze isolated adipocytes from obese mice and observed that these cells co-
express Tlr2 and TNF. Moreover, expression of these proteins was associated with insulin
resistance (394). The exact mechanisms involved in regulation of Tlr2 expression are unclear
but may be related to elevated endotoxin levels (16,20). As mentioned previously, certain
forms of LPS may interact directly with Tlr2 (387). However, recent evidence indicates that
LPS may also indirectly regulate Tlr2 through a Tlr4-specific pathway (20). These studies
insulin resistance.
revealed the presence of acyl-linked SFAs (395). Furthermore, these acylated FAs were
Kitchens and colleagues (397) reported that deacylated LPS (dLPS) antagonized LPS-
induced NFB activation and TNF expression in a human monocyte cell line. Moreover,
proinflammatory response in human monocytes (398). These data suggest that FAs,
specifically SFAs, act as endogenous ligands for Tlrs. Lee et al. (399) originally reported that
SFA stimulated NFB activation and cyclooxygenase-2 (COX2) activity through a Tlr4-
Tlr4 signaling that included activation of MyD88, IRAK-1, TRAF6, and PI3K-Akt pathways.
However, Lee and colleagues (26) demonstrated that SFA increased COX2 promoter activity
inflammatory response stimulated dimerization of Tlr2 with its co-receptors Tlr1 or Tlr6
(26). More recently, Nguyen et al. (401) demonstrated that FFAs mediated IL-6, TNF, and
MCP-1 gene expression in macrophages through Tlr2 and Tlr4 activation of JNK. Therefore,
these data demonstrate the importance of both innate immune receptors in FA-induced
inflammation.
this promotes Tlr signaling in insulin sensitive tissues, such as skeletal muscle and AT. For
instance, Reyna and colleagues (402) showed FFA concentration was associated with an
increase in Tlr4 gene and protein expression. Moreover, there was a strong correlation
between insulin resistance and Tlr4 expression in obese and diabetic subjects (402).
Alternatively, Senn (403) demonstrated that palmitate stimulated Tlr2 signaling in C2C12
myotubes, leading to activation of the proinflammatory mediators, JNK and NFB, as well as
reduced insulin-stimulated glucose uptake. Additionally, there is evidence that SFAs mediate
inflammation and insulin resistance in AT through Tlr-dependent mechanisms. Shi et al. (25)
observed that FFA-induced TNF and IL-6 expression in adipocytes was attenuated in Tlr4
knockout mice. Furthermore, these mice were protected from FFA-induced NFB activation
51
and impaired insulin signaling in AT (25). Additional studies (376-378) confirmed that Tlr4
deficient mice exhibited greater insulin sensitivity than their wild type counterparts, and that
several studies (24,401,404) suggested that SFA signaling through Tlr4 is a potential
mechanism involved in this process. Suganami and colleagues (404) utilized a co-culture
system of adipocytes and macrophages to determine that FFA released from hypertrophied
expression in macrophages, and subsequent FFA release from adipocytes. These studies
indicate that FFA released from hypertrophied adipocytes act in a paracrine and autocrine
subsequently cause local inflammation and insulin resistance. Moreover, FFAs released from
AT can then interact with Tlrs expressed in peripheral tissues, including skeletal muscle and
As discussed previously, certain MUFA and PUFA have been shown to exert anti-
inflammatory properties both in vitro and in vivo (225-229). One potential explanation for
these effects may be through antagonism of Tlr2 and Tlr4 signaling. Several investigations
(26,399,400) reported that the PUFAs, DHA and EPA, antagonized SFA-induced Tlr
signaling in RAW 264.7 macrophages. Moreover, Lee and colleagues (405) demonstrated
that monocytes isolated from subjects fed 9 to 15 g fish oil (rich in omega-3 FA) per day for
8 weeks exhibited a reduction in Tlr2 and Tlr4 agonist-mediated COX2 expression and
NFB activation. Overall, these studies indicate that SFAs act as endogenous ligands for Tlr2
52
and Tlr4, whereas PUFAs antagonize Tlr signaling, likely through a direct interaction with
these receptors.
Summary
The prevalence of obesity continues to rise dramatically in the U.S. and worldwide.
CVD. The underlying mechanism linking obesity to these diseases is the development of
increased in obesity and induce insulin resistance through a variety of mechanisms including
proinflammatory cytokines into the circulation. These inflammatory mediators can then act
endothelial cells, and neighboring adipocytes. These events promote recruitment of immune
cells into AT and further amplify the release of inflammatory mediators from AT, thus
contributing to inflammation in other insulin-sensitive tissues (i.e., liver and skeletal muscle),
resulting in subsequent insulin resistance. The ability of FFA, more specifically SFA, to
induce inflammation and insulin resistance may be dependent on the presence of specific
innate immune receptors. Tlrs recognize specific PAMPs on invading pathogens, initiating a
proinflammatory response to aid in host defense. More recently, Tlr2 and Tlr4 were observed
Furthermore, elucidating the role of Tlr2 and Tlr4 in AT inflammation will provide greater
insight into the development of obesity-related co-morbidities and help identify novel
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86
Abstract
utilized male Tlr4-deficient 10ScN mice and 10J controls. Mice were fed either low fat (low-
fat control (LFC)), high unsaturated fat (high-fat control (HFC)), or high saturated fat +
palmitate (HFP) diets ad libitum for 16 weeks. Relative to the LFC diet, the HFC diet
resulted in greater epididymal fat pad weights and adipocyte hypertrophy in both Tlr4-
deficient and normal mice. However, the 10ScN mice were completely protected against the
obesigenic effects of the HFP diet. Moreover, macrophage infiltration and monocyte
chemotactic protein-1 transcript abundance were lower in adipose tissue of 10ScN mice fed
the HFP diet, and the hyperinsulinemic response was negated. Tlr4-deficient mice also had
markedly lower circulating concentration of MCP-1 and much less nuclear factor-B protein
in nuclear extracts prepared from adipose tissue, irrespective of diet. In contrast, Tlr4
deficiency did not attenuate the induction of tumor necrosis factor- or interleukin-6
expression in adipose tissue. These data indicate that Tlr4 deficiency selectively protects
Introduction
(10). Although the mechanisms underlying the onset and progression of this inflammatory
state remain somewhat ambiguous, two recent findings have strongly implicated the adipose
tissue itself as a major contributor. First, genetic and diet-induced obesity models have not
(1,3,11,12), but also a marked adipose-specific infiltration of macrophages that show gene
expression profiles consistent with inflammation (13-15). Secondly, the adipocyte itself is
proinflammatory gene expression and the release of these cytokines from cultured adipocytes
(16-18). Furthermore, both Tlr2 and Tlr4 expression are increased in adipose tissue in
It is clear from multiple obesity models that adipocytes and macrophages, localized in
adipose tissue, produce proinflammatory mediators, the mechanistic links among adipocyte
hypertrophy, dietary fat, inflammation, and macrophage recruitment and activation have not
been delineated. However, direct regulation of Tlr4 by certain saturated fatty acids (SFA) is
a likely component of this process because palmitate and other SFA act directly to stimulate
macrophages (20,21). Furthermore, Suganami and colleagues (21) recently determined that
free fatty acids (FFA) released from hypertrophic adipocytes can signal macrophages through
Tlr4 and stimulate release of tumor necrosis factor (TNF). Additional studies by other
groups (20,22-24) have shown that Tlr4 deficient mice are less susceptible to fat-induced
88
inflammation and insulin resistance, and that C3H/HeJ mice are protected against
hyperglycemia and inflammation in adipose tissue when Tlr4 signaling is blunted (22).
inflammation in adipose tissue seems quite clear, whether this receptor contributes to the
onset and progression of obesity is controversial. Elegant in vivo studies with a Tlr4
knockout mouse model have shown blunted inflammation, yet a greater adiposity in females
(23). In contrast, Tsukomo et al. (25) recently reported evidence that a loss of function
mutation in Tlr4 protects against high fat, diet-induced obesity. Albeit, there is currently a
paucity of data that address the possibility that dietary SFA vs. unsaturated fatty acids (UFA)
influence the ability of Tlr4 to promote adipose accretion. Consequently, we used the 10ScN
mouse strain, which has a 74 kb deletion from chromosome 4 that precludes expression and
production of Tlr4, to test the hypothesis that Tlr4 specifically mediates obesity and
Animals and animal care. Male Tlr4 deficient C57BL/10ScN mice (Cat No. 003752)
derived from the C57BL/10 subline and corresponding control male C57BL/10J mice (Cat
No. 000665), were obtained from Jackson Laboratories (Bar Harbor, ME) at 6-8 weeks of
age. Subsequently, lack of Tlr4 mRNA was confirmed with real time PCR. Animals were
automatically controlled 12-hour light:dark cycle. Mice were acclimated to the environment
and provided unlimited access to food and water. Animals from each genotype were then
control (LFC), high fat control (HFC), or high fat palmitate (HFP) for 16 weeks. Both high
89
fat diets were semi-purified, powdered diets based on American Institute of Nutrition
recommendations (26) that were modified to induce obesity by providing 60% of calories
from lipid (Table 1). The high fat diets were identical except the source of fat used was either
soybean oil in the HFC (Harlan Teklad) or a mixture of lard (Harlan Teklad) and purified
palmitate (Nu-Check Prep, Inc.) in the HFP. The LFC diet contained 12% of calories from
lipid. Food intake was measured daily and used to calculate total energy intake. Mice were
fasted overnight (6-8 h) and euthanized by CO2 asphyxiation for all blood and tissue
collections. Blood was collected by heart puncture, placed on ice, and serum was collected
by centrifugation and frozen at -80oC until analyzed. Animals were weighed and abdominal
fat pads and liver were removed and weighed. All extracted tissues were immediately frozen
in liquid nitrogen and stored at -80C prior to RNA extraction and immuno-histochemical
analysis. All experimental protocols for animal care and use were approved by the
Institutional Animal Care and Use Committee at Iowa State University, Ames, IA, USA.
insulin (Linco) were measured using commercial assay kits. Leptin, monocyte chemotactic
protein-1 (MCP-1), adiponectin (ADN), and interleukin-6 (IL-6) serum concentrations were
measured using ELISA kits (R&D Systems). Serum FFA was measured with NEFA-C
Quantitative real-time PCR. Total RNA was extracted from frozen adipose tissue
DNase contamination was removed with DNase-freeTM (Ambion, Inc.). First-strand cDNA
was synthesized using SuperScript III First-Strand Synthesis System for RT-PCR
(Invitrogen Corp.). Primer sequences for mouse sense and anti-sense primers are listed in
90
Table 2. Thermal cycling conditions for PCR reactions were 95C for 3 min followed by 40
cycles of 95C for 15 s, 60C for 30 s, and 72C for 30 s. Polymerase reaction products
amplified by these primers were cloned into pGEMT vector (Promega) and sequenced for
verification. Real-time reactions were carried out on an iCycler real time machine (BioRad)
using the IQTM SYBR Green Supermix kit (BioRad). The abundance of each gene product
was calculated by regressing against the standard curve generated in the same reaction with
their respective plasmid. All genes of interest were normalized to beta actin and expressed as
temperature in 10% zinc-formalin solution and embedded in paraffin. Five micron sections
were cut at 50 m intervals and mounted on glass slides, deparaffinized in xylene, and
stained for expression of F4/80 with monoclonal antibody (Serotec) as previously described
(14). For each mouse, four different fields were selected and adipocyte area was determined
using AxioVision v4.5.0.0 (Carl Zeiss, Germany). F4/80-positive macrophages were then
counted at 40X magnification and divided by total cell number to obtain percent F4/80-
positive cells.
Tissue and plasma fatty acid profile. Lipids from adipose and serum samples were
extracted by the method of Lepage and Roy (27) with minor modifications. Briefly, 0.5 g of
tissue was homogenized in 2.5 mL 4:1 methanol:hexane and 200 L of 3.7 mmol
heptadecanoic acid/L methanol was added to each sample as an internal standard. Fatty acid
(Hewlett-Packard, Palo Alto, CA) fitted with an Omegawax 320 (30 m x 0.32 mm ID, 0.25
m) capillary column (Sigma-Aldrich, St Louis, MO USA). Hydrogen was the carrier gas.
91
The temperature program ranged from 80C to 250C with a temperature rise of 5C/min.
The injector and detector temperatures were 250C and 1 L of sample was injected and run
splitless. Fatty acids were identified by their retention times on the column with respect to
appropriate standards.
EMSA and Activated NFB p65 ELISA. Adipose tissue nuclear extracts and mobility
shift assay were prepared and validated as previously described (16). The consensus NF-B
polynucleotide kinase (Santa Cruz). Binding of nuclear proteins to the labeled probe was
done by incubating 50 g nuclear proteins with 50,000 cpm of labeled probe for 30 min at
reaction volume of 40 L. For quantitative NFB p65 DNA binding, 10g of nuclear protein
was assayed for the presence of activated p65 by ELISA using antibodies specific for
activated p65 following binding to NFB consensus sequence (TransAM Active Motif).
Statistical analyses. Data were tested for normality and analyzed using the mixed-
model analysis with diet and genotype considered fixed effects, and the time dietary
main effects are indicated as Diet*Genotype effects. All indicated P-values were two-tailed
and Bonferroni corrected. Differences were considered significant at P < 0.05 and a tendency
at P < 0.10. Values are presented as least squares (LS) means SEM.
Results
diet high in saturated fat, without reducing caloric intake. We examined whether the fatty
92
acid composition of the diet influenced the obesigenic effect of high fat diets in male Tlr4
mutant (10ScN) and normal (10J) mice. Regardless of diet, 10ScN mice weighed on average
3.8 g less than 10J mice after 16 weeks on the experimental diets (Genotype, P<0.0001,
Figure 3A). Furthermore, the HFC diet increased body weight in both genotypes (Diet,
P<0.0001), but average weekly energy intake (Figure 3B) was reduced by the HFC diet
(Diet, P<0.0001). Additionally, epididymal fat pad weight was increased by the HFC diet in
both genotypes. However, only the mice lacking Tlr4 were protected against the obesigenic
effect of the HFP diet (Figure 3C, Diet*Genotype, P=0.05). Similar results were obtained for
epididymal adipocyte size (Figure 3D) in that all mice fed the HFC diet had larger cells, but
only the normal strain fed the HFP diet had enlarged cells vs. the LFC (Diet*Genotype,
P=0.05).
we measured SFA, n-3, and n-6 fatty acid contents (Table 3). As expected, relative to the
LFC and HFP diets, both palmitate and total SFA were reduced in mice fed the HFC diet
(Diet, P<0.002), and n-3 fatty acids (docosahexaenoic acid and eicosapentaenoic acid) were
lower in mice fed the HFP diet (Diet, P<0.002). Furthermore, the n-6 fatty acid content was
higher in mutant mice (Genotype, P<0.005) and was also reduced by the HFP diet (Diet,
P<0.03). However, the effects of diet and genotype on n-3 and n-6 fatty acid contents were
not sufficient to alter the n-6 to n-3 ratio in this particular adipose depot.
insight into the effect of high fat intake on insulin sensitivity in Tlr4 deficient mice, we
measured fasting serum glucose (Figure 4A) and insulin (Figure 4B) following 16 weeks on
experimental diets. Glucose concentrations were lower in the mutant strain (Genotype,
93
P=0.0068), and were reduced in both genotypes fed either high fat diet (Diet, P=0.0014).
Insulin concentrations were also lower in Tlr4 mutant mice (Genotype, P<0.0001).
Alternatively, the HFC diet nearly doubled serum insulin in these mice, but this response
only approached significance (Diet, P=0.111), and there was no effect on the normal strain
ratios (GIR) in mutant mice fed the HFC diet as compared with the LFC or HFP diets, and
the normal 10J mice had similar GIR, regardless of diet (Diet*Genotype, P=0.028).
We then measured several markers of inflammation that have been associated with
obesity. The serum concentration of leptin was greater in all mice fed the HFC diet (Diet,
P=0.0004, Figure 4D), and serum adiponectin was lower in the mutant strain (Genotype,
P=0.043, Figure 4E). Additionally, the HFC diet increased serum adiponectin relative to the
control and HFP diets, but only in the mutant strain (Genotype*Diet, P=0.031). Serum MCP-
1 was seven-fold higher in 10J vs. mutant mice (Genotype, P<0.0001, Figure 4F), but was
dietary SFA vs. UFA, and was partly ameliorated by Tlr4 deficiency. To determine if
infiltration and measured several inflammatory markers in adipose tissue. The percentage of
F4/80-positive cells (i.e., macrophages) in adipose tissue of 10J control mice was greater in
mice fed either HF diet (Diet P<0.0001, Figure 5A). Alternatively, Tlr4 deficiency
positive cells was associated with the formation of distinct crown-like structures (CLS)
(Figure 4B).
and cytokines. MCP-1 expression was increased in the epididymal adipose tissue of 10J mice
fed the high fat diets (Genotype*Diet, P=0.021, Figure 6A). There was also a trend for
increased CCR2 transcript abundance in mice fed either HF diet (Diet, P=0.089, Figure 6B).
Expression of the proinflammatory cytokines, TNF (Figure 6C) and IL-6 (Figure 6D), were
increased by over 2- and 3-fold in adipose tissue of mice fed the HFP diet (Diet, P=0.013 and
P<0.0001, respectively). Furthermore, the magnitude of the increase in IL-6 was greater in
mutant vs. normal mice fed the HFP diet (Genotype*Diet, P=0.074). Tlr2 expression was
increased over 2-fold with diet-induced obesity, irrespective of genotype (Diet Effect,
qualitative assessment of NFB translocation. There was a marked reduction in the nuclear
quantitative results were obtained using a NFB p65 DNA binding ELISA, which indicated
adipose tissue nuclear NFB concentration in mutant mice was 50% less than that in 10J
Discussion
The data presented herein establish a novel relationship between obesity and Tlr4.
The absence of a functional Tlr4 due to the 74 kb deletion on chromosome 4 in the 10ScN
strain selectively protects against the obesigenic effect of a diet high in SFA, but not that of
95
an isocaloric diet high in UFA in male 10ScN mice. Although we did not quantify total body
fat mass in this study, adipocyte size mirrored epididymal fat pad weights, and thus reinforce
our conclusion that the absence of Tlr4 protected against diet-induced obesity (DIO), but
only when the diet was based on SFA. It is also important to note that this resistance to
and is thus of metabolic origin, rather than a simple limitation in energy intake. These
findings may implicate energy expenditure, and this possibility and potential underlying
findings indicated that the gut microbial population influences adipose accretion through
effects on energy availability and regulation of the fasting-induced adipocyte factor (Fiaf).
The Fiaf protein is a circulating lipoprotein lipase inhibitor expressed by the intestinal
epithelium and adipocyte, and knockout models have shown that the absence of Fiaf
microbiota in germ-free mice (28). Consequently, it seems possible that the absence of Tlr4
precludes changes in the gut microbiota in response to SFA that would alter Fiaf expression
Recent reports (22-25) have indicated mixed results with regards to DIO in Tlr4
mutant or knockout mice. Our results are consistent with those of Tsukumo et al. (25), who
also used males, but from the C3H/HeJ model. In contrast, Shi et al. (23) used females from
a Tlr4 knockout model backcrossed with C57BL6J for six generations, and determined that
these mice succumbed to DIO when fed a high-fat diet containing lard. Furthermore, Poggi et
al. (24) reported heavier epididymal adipose weights and adipocyte hypertrophy in male
C3H/HeJ mice consuming a high fat diet based on anhydrous milk fat, which likely varies
96
considerably in fatty acid composition. Although the mechanisms underlying these disparate
results are not yet apparent, Tsukumo et al. (25) suggested that metabolic and physiologic
differences relating to strain and background are likely to be important factors, and our data
clearly indicate a strong linkage between Tlr4 function, dietary fat source, and DIO. Equally
important, Cani et al. (29) provided convincing evidence that mice consuming a high fat diet
containing both corn oil and lard had increased circulating concentration of bacterial
lipopolysaccharide that triggered obesity and inflammation. Because mice lacking the CD14
co-receptor for Tlr4, which precludes formation of a functional Tlr4 complex, were resistant
to this phenomenon, these researchers suggested that endotoxemia facilitated by dietary fat
intake largely mediates the effects of a high fat diet on obesity and inflammation via a Tlr4-
mediated signaling pathway. Collectively, the data substantiate the possibility that SFA
promote obesity post-absorptively by direct activation of Tlr4, and (or) promote the uptake of
utmost importance in the future to establish the effects of specific Tlr4 mutations and
knockout models on obesity with respect to specific dietary fat sources and fatty acid
profiles.
associated with the onset and progression of insulin resistance, and ultimately the transition
to frank diabetes. We have shown previously that treating cultured adipocytes with palmitate
results in activation of NFB and proinflammatory cytokine expression and release into the
culture media (30). Furthermore, using in vivo and in vitro experiments, Shi et al. (23)
palmitate or SFA on adipocytes. With respect to the present data, several points are notable.
97
First of all, the only inflammatory marker attenuated by the absence of Tlr4 was MCP-1.
Whereas the high fat diets, irrespective of whether saturated or unsaturated in composition,
resulted in increased MCP-1 expression in epididymal adipose tissue of normal mice, neither
diet altered expression in mutant mice. Furthermore, the expression of both IL-6 and TNF
were markedly increased by the HFP diet, irrespective of genotype, and chemokine (C-C
motif) receptor 2 (CCR2) expression was increased by the high fat diets in both genotypes.
These results indicate not only that some inflammatory responses to high fat diets may be
mediated independently of Tlr4, at least at the mRNA level, but also that some responses to
high fat intake occur whether the fat is comprised predominantly of SFA or UFA. It should
also be noted that even in the absence of epididymal adipose expansion and adipocyte
hypertrophy in the mutant mice fed the HFP diet, both IL-6 and TNF expression were
fatty acyl Co-A synthase attenuates the activation of NFB by palmitate, but exacerbates IL-
6 release into the culture media (30). Finally, the absence of a functional Tlr4 not only
precluded the adipocyte hypertrophy in mice fed the HFP diet, but also blocked infiltration of
adipose tissue with macrophages. This is consistent with the hypothesis that it is adipocyte
hypertrophy that ultimately signals macrophage recruitment, and it is clear that this response
The serum MCP-1 and adipose NFB data show a striking effect of genotype in that
both were markedly depressed in the mutant mice, and were largely unresponsive to diet.
Although serum MCP-1 concentration was barely measurable in mutant mice, MCP-1
adipose tissue expression differed little from that of normal mice. This perhaps indicated
98
that adipose tissue contributed little to circulating MCP-1 in these mice, but we cannot
discount the possibility that the adipose concentration was sufficient to be of physiological
quantitative assessments indicated that the nuclear content of this important transcription
factor is significantly lower in mutant vs. normal mice with little effect of diet. We have
shown previously that the transcriptional activity of NFB in adipocytes can be uncoupled
from proinflammatory cytokine responses (30), but it is perhaps important that Shi et al. (23)
In summary, our study indicated that the Tlr4 mutation in male 10ScN mice
selectively blocked SFA-induced obesity, and that this response was associated with an
absence of adipose infiltration with macrophages and improved markers of insulin sensitivity
as reflected in the lower ratios of glucose to insulin. These results point to a unique
relationship between Tlr4 and saturated fatty acids, and underscore the potential for
interactions between specific dietary fatty acids and Tlr4 mutations to impact the
Acknowledgements
This work was supported by a grant from the Center for Designing Foods to Improve
Nutrition (CDFIN), and Nutrition and Wellness Research Center at Iowa State University.
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Table 3. Adipose tissue fatty acid profiles in normal and Tlr4 mutant mice fed low fat
control, high fat control, and high fat-palmitate diets for 16 weeks
Diet Genotype n Palmitate SFA n3 FA n6 FA n6:n3 Ratio
________________________g/100g________________________
LFCa 10ScN 6 31.35 3.47 33.68 3.38 0.49 1.00 41.56 6.19 60.40 28.22
HFCa 10ScN 6 18.10 3.22 19.84 3.16 2.23 0.98 49.51 5.58 77.63 27.05
HFPa 10ScN 6 28.45 3.18 30.61 3.10 n.d. 36.59 5.63 85.99 26.36
LFCa 10J 6 24.02 2.94 26.53 2.88 2.17 0.95 31.05 5.18 27.88 24.67
HFCa 10J 6 17.64 3.13 19.75 3.06 2.16 0.96 32.42 5.57 66.68 25.77
HFPa 10J 6 29.76 3.19 32.15 3.12 0.53 0.99 15.18 5.53 35.62 26.92
Main Effects
Genotype P = 0.48 P = 0.52 P = 0.18 P = 0.0055 P = 0.48
Diet P = 0.0026 P = 0.0014 P = 0.0025 P = 0.033 P = 0.21
Interaction
Diet*Genotype P = 0.32 P = 0.29 P = 0.22 P = 0.61 P = 0.67
a
Low Fat Control (LFC), High Fat Control (HFC), and High Fat Palmitate (HFP) diets
105
Figure 3. Tlr4 deficiency protects mice from increased adiposity due to consumption of a
diet high in saturated fat.
All measures were taken after 16 weeks on experimental diets (n = 25 unless noted
otherwise). (A) Body weight of 10ScN (white bars) and 10J (black bars) mice (Diet effect
P<0.0001, Genotype effect, P<0.0001, Diet*Genotype interaction P=0.84). (B) Average
weekly energy intake (kcal/week) of 10ScN and 10J mice (Diet effect P<0.0001, Genotype
effect P=0.34, Diet*Genotype interaction P=0.94). (C) Epididymal fat pad weight of 10ScN
and 10J mice (Diet effect P<0.0001, Genotype effect P<0.0001, Diet*Genotype interaction
P=0.058). (D) Adipocyte size (m2) of 10ScN and 10J mice (n = 6, Diet effect P=0.0079,
Genotype effect P<0.0001, Diet*Genotype interaction P=0.052). All displayed values
represent LS means SEM for Diet*Genotype interaction. When interaction term was
P<0.15, Bonferroni correction was used to determine significant difference between means as
indicated by different letters. Significant main effects (Genotype and Diet) were also
indicated by * (Genotype effect) and # (Diet effect) over LS means.
106
Figure 4. Tlr4 deficiency improved markers of insulin sensitivity and inflammation in mice
fed a diet high in saturated fat.
All measures were taken after 16 weeks on experimental diets (n = 10 unless noted
otherwise). (A) Serum glucose (Diet effect P=0.0014, Genotype effect P=0.0068,
Diet*Genotype interaction P=0.57), (B) serum insulin (Diet effect P=0.111, Genotype effect
P<0.0001, Diet*Genotype interaction P=0.15), (C) glucose to insulin ratio (Diet effect
P=0.038, Genotype effect P<0.0001, Diet*Genotype interaction P=0.028), (D) serum leptin
(Diet effect P=0.0004, Genotype effect P=0.50, Diet*Genotype interaction P=0.15), (E)
serum adiponectin (Diet effect P=0.25, Genotype effect P=0.043, Diet*Genotype interaction
P=0.11), and (F) serum MCP-1 (Diet effect P=0.96, Genotype effect P<0.0001,
Diet*Genotype interaction P=0.78) were measured in 10ScN (white bars) and 10J (black
bars) mice following 16 weeks on diet. All displayed values represent LS means SEM for
Diet*Genotype interaction. When interaction term was P<0.15, Bonferroni correction was
used to determine significant difference between means as indicated by different letters.
Significant main effects (genotype and diet) were also indicated by * (genotype effect) and #
(diet effect) over LS means.
107
Five micron sections were cut at 50m intervals and mounted on glass slides, deparaffinized
in xylene, and stained for expression of F4/80 with monoclonal antibody. Adipose sections
were visualized under 40X magnification. (A) Percent F4/80 positive cells (n = 5, genotype
effect P=0.65, diet effect P=0<0.0001, genotype*diet interaction P=0.0002), were measured
in 10ScN (white bars) and 10J (black bars) mice following 16 weeks on diet. All displayed
values represent LS means SEM for Diet*Genotype interaction. When interaction term was
P<0.15, Bonferroni correction was used to determine significant difference between means as
indicated by different letters. Significant main effects (genotype and diet) were also indicated
by * (genotype effect) and # (diet effect) over LS means.
(B) Representative images from 10ScN and 10J mice fed low fat (LFC), high fat (HFC), or
HFP diet.
108
Figure 6. Adipose tissue inflammatory gene expression in Tlr4 deficient (10ScN) mice is
differentially regulated in response to the HFP diet.
All measures were taken after 16 weeks on experimental diets (n = 6 unless noted otherwise).
(A) MCP-1 (Diet effect P=0.70, Genotype effect P=0.31, Diet*Genotype interaction
P=0.021), (B) CCR2 (Diet effect P=0.089, Genotype effect P=0.33, Diet*Genotype
interaction P=0.35), (C) TNF (Diet effect P=0. 013, Genotype effect P=0.56,
Diet*Genotype interaction P=0.93), (D) IL-6 (Diet effect P<0.0001, Genotype effect P=0.98,
Diet*Genotype interaction P=0.074), and (E) Tlr2 (Diet effect P=0. 035, Genotype effect
P=0.45, Diet*Genotype interaction P=0.65) were measured in 10ScN (white bars) and 10J
(black bars) mice following 16 weeks on diet. All displayed values represent LS means
SEM for Diet*Genotype interaction. When interaction term was P<0.15, Bonferroni
correction was used to determine significant difference between means as indicated by
different letters. Significant main effects (genotype and diet) were also indicated by *
(genotype effect) and # (diet effect) over LS means.
109
Figure 7. Tlr4 deficiency attenuates NFB activation in adipose tissue of Tlr4 deficient
(10ScN) mice.
Animals were fasted 6-8h and epididymal fat pad was collected. Nuclear extracts were
obtained from adipose tissue and subsequently used for (A) NFB EMSA, each lane
represents an individual sample (n = 2). (B) NFB p65 DNA binding ELISA. Nuclear
extracts were used to quantify NFB DNA binding; 10ScN (white bars) and 10J (black bars)
(n = 4, Diet effect P=0.39, Genotype effect P=0.0015, Diet*Genotype interaction P=0.43).
All displayed values represent LS means SEM for Diet*Genotype interaction. When
interaction term was P<0.15, Bonferroni correction was used to determine significant
difference between means as indicated by different letters. Significant main effects (genotype
and diet) were also indicated by * (genotype effect) and # (diet effect) over LS means.
110
Abstract
Saturated fatty acids (SFAs) are known to induce inflammation and insulin resistance
in adipocytes through toll-like receptor-4 (Tlr4) signaling, but the mechanisms are not well
delineated. Furthermore, the potential roles of Tlr2 and the c-jun N-terminal kinase (JNK) in
lipopolysaccharide (LPS), and the toll-like receptor-2 (Tlr2) agonist, zymosan A (ZymA),
Corresponding with the reduction of insulin sensitivity was an increased expression of IL-6,
(NFB) and activator protein-1 (AP-1). Reactive oxygen species (ROS) accumulation was
also observed in palmitate and Tlr agonist treated adipocytes. The JNK inhibitor, SP600125,
attenuated insulin resistance mediated by SFA and Tlr agonists, which corresponded with a
the receptor as a potential target for SFA. Moreover, activation of JNK also appeared to be
essential to Tlr2-, as well as Tlr4-, induced insulin resistance and oxidative stress.
Introduction
Adipose tissue (AT) from obese individuals shows activation of several distinct
inflammatory pathways, including those of NFB and JNK (1,2). These proinflammatory
signals stimulate production of cytokines and chemokines, including interleukin-6 (IL-6) and
111
humans is associated with insulin resistance and an increased risk of diabetes mellitus (DM)
(1,2,5). These strong associations between obesity, inflammation, and insulin resistance
Saturated fatty acids (SFA) activate inflammatory pathways and cause insulin resistance in
myocytes (6-9), macrophages (10), and adipocytes (11). Furthermore, increased lipolytic
(NEFAs), which can augment the inflammatory response of AT through the activation of
neighboring macrophages and adipocytes (10). NEFAs are also implicated in the production
of ROS and the ensuing oxidative stress (12). In the obese state, this increased flux of NEFA
stress, and insulin resistance may involve several distinct pathways. In primary myocytes
and hepatocytes, NFB and AP-1 signaling are critical (9,15). Tlr4 activation was also
ligand for Tlr4, induces inflammation through JNK/AP-1 and NFB signaling pathways in
several cell types (21-25). Recently, Shi and colleagues (3) reported an attenuation of lipid-
induced NFB activation and insulin resistance in AT of Tlr4 knockout mice. Several
additional studies (18,19,26,27) have confirmed that Tlr4 deficiency protects animals from
that Tlr4 deficient 10ScN mice, were specifically protected from SFA-induced adiposity and
that they have reduced macrophage accumulation in AT (28). In contrast, the pro-
112
inflammatory cytokines IL-6 and TNF were elevated in AT of 10ScN mice fed the same
SFA-diet. These data suggest that whereas Tlr4 signaling plays a considerable role in SFA-
also exist.
Like Tlr4, the Tlr2 receptor is increased in AT of obese and diabetic individuals
(22,29). Adipocytes of mice fed a high fat diet have greater expression of this pattern
recognition receptor (30). It is known that SFA signal through Tlr2 and activate IKK/NFB
and JNK/AP-1 pathways in myotubes and epithelial cells, respectively (31-33). Additionally,
Senn (31) reported that palmitate-induced insulin resistance in C2C12 myotubes is attenuated
accumulation after vascular injury is also blunted in Tlr2 knockout mice (34). Thus,
considering the recognized involvement of Tlr2 in oxidative stress and insulin resistance in
multiple cell types, we hypothesized that Tlr2 agonists Zymosan A (ZymA) and palmitate
would activate inflammatory signaling in the adipocyte and thereby promote inflammation,
The data presented herein indicated clearly that ZymA and palmitate activate JNK
and cause NFB and AP-1 nuclear translocation and proinflammatory cytokine production.
Furthermore, inhibition of JNK blocked Tlr2 and Tlr4 agonist-induced ROS and insulin
resistance in adipocytes. These data also provide novel evidence supporting a potential role
Cells and Reagents. 3T3-L1 preadipocytes were obtained from American Type
Culture Collection (Manassas, VA, USA). All reagents were obtained from Sigma-Aldrich
Cell Culture and Treatment. 3T3-L1 preadipocytes were grown in 5% CO2 in low
glucose (5.5 mM) DMEM containing 10% fetal bovine serum (FBS; Atlanta Biologicals,
post-confluence (0 d), cells were induced to differentiate with medium containing 10% FBS,
for 48 h. Thereafter, fresh growth media supplemented with 1.7 M insulin was added for 2-
3 d followed by insulin-free growth media for the remaining culture period. At 10-12 d, cells
were cultured in medium containing 10% FBS, 0.3 M bovine serum albumin (BSA), and
0.1% methanol with one or a combination of the following treatments: 0.5 mM sodium
palmitate, 10 g/mL LPS from Escherichia coli 055:B5, 20 g/mL ZymA from
Quantitative real-time PCR. Total RNA was extracted from cells using a
Potential DNase contamination was removed with DNase-freeTM (Ambion, Foster City, CA,
USA). First-strand cDNA was synthesized using SuperScript III First-Strand Synthesis
System for RT-PCR (Invitrogen, Carlsbad, CA, USA). Primer sequences used to detect -
actin, Tlr4, Tlr2, MCP-1, and IL-6 are described elsewhere (28). Thermal cycling conditions
for PCR reactions were 95C for 3 min followed by 40 cycles of 95C for 15 s, 60C for 30
114
s, and 72C for 30 s. Products amplified by these primers were cloned into pGEMT vector
(Promega, Madison, WI, USA) and sequenced to confirm transcript identity. Real-time PCR
reactions were carried out with an iCycler using the IQTM SYBR Green Super Mix kit (Bio-
Rad, Hercules, CA, USA). The abundance of each transcript was calculated by regression
against the standard curve generated on the same plate with the respective plasmid.
Expression levels of genes of interest were normalized to -actin and are reported as fold
were determined using commercially available ELISA kits (Biovision, Mountain View, CA,
USA and R&D Systems, Minneapolis, MN, USA, respectively). Glycerol release was
Western Blot Analysis. Cells were grown in 6 well tissue culture plates, and treated as
described above. To harvest protein for analysis, media from cells was aspirated, and cells
were rinsed once with ice cold PBS. Lysis buffer (10 mM Hepes pH 2.5, 20 mM Tris-HCl
g/mL aprotinin, 1 g/mL pepstatin, and 1 mM PMSF) was added to each well of cells,
which were then scraped from the plate. Cell protein lysates were resolved by electrophoresis
western blot analyses. The membrane was blocked with 5% skim milk in tris buffered saline
overnight at 4C. After washing three times with TBST (0.05% Tween 20, 50 mM TrisHCl,
115
pH 7.5, and 150 mM NaCl) for 15 min, the membrane was incubated for 1 h with anti-rabbit
IgG secondary antibody conjugated with horseradish peroxidase. The signal was detected
using the Phototope-HRP Western Blot Detection System (Cell Signaling Technology,
NFB and AP-1 Electrophoretic Mobility Shift Assay (EMSA). Nuclear extracts for
mobility shift assay reagents were prepared and validated as previously described (36).
Briefly, the consensus NF-B and AP-1 oligonucleotides (Santa Cruz, CA, USA) were end-
labeled with [-32P] ATP (PerkinElmer, Waltham, MA, USA) using T4 polynucleotide kinase
(Santa Cruz, Santa Cruz, CA, USA). 10-30 g of nuclear proteins were incubated with
50,000 cpm of labeled probe for 30 min at room temperature in binding buffer [2 mM
HEPES, 50 mM KCl, 2 mM EDTA, 10% glycerol, and 1% BSA (wt/vol)] with 2 g poly(dI-
Invitrogen, Carlsbad, CA, USA) was added to 1X Krebs Ringer Buffer (KRB; 12 mM
HEPES, pH 7.4, 121 mM NaCl, 4.9 mM KCl, 1.2 mM MgSO4, 0.33 mM CaCl2). Cells were
then incubated for 45 min at 37C, after which medium was aspirated and cells washed three
times with warm phosphate buffered saline (PBS). Culture plates were immediately read
using a fluorescent plate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA)
with 435 nm excitation and 550 nm emission wavelengths, respectively. Fluorescence was
normalized to total protein as determined with the bicinchoninic acid assay (BCA; Pierce,
cytochalasin B and with 100 nM insulin for 30 or 10 min at 37C, respectively, in 1X Krebs
Ringer buffer (KRB). Non-insulin stimulated control cells were treated identically. Cell
media was then supplemented with 0.5 Ci/mL [3-H]-2-DG and 120 M non-labeled 2-DG
and cells were incubated for an additional 25 min. Glucose uptake was terminated by
aspiration of media. Thereafter, the cells were washed with 1X KRB and lysed with 0.1 N
NaOH. Cell lysates were used to determine 2-DG uptake via scintillation counting. Uptake
was normalized to total lysate protein as determined by BCA assay. Data are presented as
Statistical analyses. Data were tested for normality and analyzed using the mixed-
model analysis. Treatment was considered the fixed effect and experimental repetition the
random effect. The main effects (treatment and replicate) were tested against the treatment x
replicate interaction term. When protected by a significant F-test, mean separation was
accomplished using the least-squares mean separation (pdiff) procedure. Differences were
Results
Time dependent induction of insulin resistance by palmitate, ZymA, and LPS in 3T3-
L1 adipocytes. Based on previous data, we investigated the ability of LPS and palmitate
compounds (at previously studied doses) to mediate insulin resistance in a time dependent
insulin resistance.
that caused by palmitate and (or) LPS. As shown in Table 4, the Tlr2 agonist, ZymA also
caused a 2-fold increase in IL-6 gene expression (P=0.05) which corresponded with a greater
than 10-fold increase in media IL-6 content (P<0.0001, Table 4). Consistent with prior
(P<0.0001) and 30-fold increase in IL-6 media content (P<0.0001). We further evaluated the
chemokine, MCP-1. As with palmitate and LPS, ZymA increased MCP-1 expression vs.
control cells (P<0.0001, Table 4) with a near 2-fold greater transcript abundance. The
expression of Tlr2 and Tlr4 were evaluated to assess potential feedback regulation of Tlr
ligands on their respective receptors. Although Tlr2 expression was induced by all treatments
(P<0.0001, Table 4), Tlr4 was largely unresponsive to these pro-inflammatory mediators
(Table 4), with the exception that LPS tended to reduce expression of Tlr4 (P=0.09). To
investigate the roles that Tlr ligands play in the accumulation of ROS, we measured several
markers of oxidative stress and ROS production. First, we quantified expression of the
inducible nitric oxide synthase gene, and found that palmitate, LPS, and ZymA all caused a
5-fold increase in iNOS expression (P<0.0001, Table 5). Next, we assessed ROS
accumulation, and demonstrated that all three ligands caused a 50% increase in intracellular
ROS (P<0.01, Table 5). However, an increase in media nitrite concentrations (25%, P=0.008,
Table 5), indicative of extracellular oxidative stress, was apparent only in response to
palmitate.
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NFB, AP-1, and JNK were activated by palmitate, ZymA and LPS in adipocytes. We
next studied potential mechanisms underlying the pro-inflammatory effects of palmitate and
the classic Tlr ligands. JNK has been implicated previously in proinflammatory signaling in
kDa) and JNK2 (Tyr185, 54 kDa) as an indicator of activation in cells treated with palmitate,
LPS, and ZymA. All three pro-inflammatory mediators significantly increased JNK
phosphorylation (P<0.05, Fig. 9A-B). Activation of JNK is known to stimulate the activity of
the pro-inflammatory transcription factors, NFB and AP-1. After 48 h, there was increased
activation of NFB by palmitate, LPS, and ZymA (Fig. 9C). Consistent with previous results,
the LPS treatment had the most pronounced effect on NFB activation under the applied
experimental conditions. Likewise, AP-1 activation was also increased by all of the pro-
inflammatory mediators (Fig. 9D), and was greatest in cells exposed to ZymA.
oxidative stress, and insulin resistance in adipocytes. Our findings indicated that palmitate,
LPS, and ZymA all induced JNK activation. Hence, we sought to determine whether
inhibition of JNK would attenuate the inflammatory, oxidative, and insulin resistance
control cells, there was a 22% (P=0.045), 45% (P=0.0005), and 33% (P=0.0045) reduction in
palmitate-, LPS- and ZymA-induced JNK activation, respectively, when cells were also
incubated with the SP600125 inhibitor (Fig. 10A-B). Furthermore, as shown in Fig. 10C,
(P<0.0001), and attenuated the induction by LPS and ZymA. In contrast, inhibition of JNK
119
did not alter the effects of LPS or ZymA on MCP-1, Tlr2, or Tlr4 expression (Fig. 10D).
However, there was a small (approximately 20%), but significant, decrease in palmitate-
induced MCP-1 (P=0.023), Tlr2 (P<0.0001, Fig. 10E), and Tlr4 (P=0.0014, Fig. 10F).
were associated with changes in NFB and AP-1 activation, adipocytes were treated with
LPS, palmitate, or ZymA in the presence or absence of SP600125. The JNK inhibitor
reduced the induction of NFB by all three pro-inflammatory mediators (Fig. 10A), but this
was qualitatively more pronounced for palmitate. Conversely, inhibition of JNK only
reduced activation of AP-1 in cells stimulated with palmitate or ZymA (Fig. 10B).
JNK mediated the insulin resistance caused by palmitate and Tlr ligands. There was
only a small (non-significant) reduction in LPS- and ZymA-induced iNOS expression in the
presence of the JNK inhibitor (Fig. 10C). Conversely, palmitate-induced iNOS expression
was dramatically reduced (over 60%, P<0.0001) with inhibitor. Furthermore, ROS
(P<0.0001) for cells stimulated with palmitate or Tlr agonist. To determine if the attenuated
ROS accumulation impacted insulin sensitivity, we assessed glucose uptake using 2-DG
(Fig. 10E). Insulin-stimulated glucose uptake was improved over 40% in cells stimulated
with palmitate (P=0.0026), LPS (P=0.0018), or ZymA (P<0.0001) when JNK was blocked by
the inhibitor.
Discussion
The data presented herein indicated several important points regarding the regulation
of inflammation and insulin sensitivity in adipocytes by palmitate and Tlr ligands. First of
all, we showed that activation of Tlr2 resulted in a pro-inflammatory response and insulin
120
resistance that bore marked similarity to responses caused by palmitate or activation of Tlr4
by LPS. Palmitate and both Tlr agonists resulted in increased nuclear localization of the
NFB and AP1 transcription factors, and caused increased expression of MCP-1, Tlr2, and
iNOS, as well as increased ROS accumulation. Furthermore, as with palmitate and LPS,
activation of Tlr2 by ZymA activated JNK and attenuated the ability of insulin to stimulate
in vivo has not yet been established, but Nguyen et al. (39) recently demonstrated that NEFA
activation of JNK involves both Tlr2 and Tlr4 in RAW 264.7 macrophages. The fact that
Tlr2 is upregulated by palmitate, LPS, or ZymA, whereas Tlr4 is not, underscores the
expression of Tlr2 and thus amplification of the inflammatory response (40). Moreover, the
ability of these receptors to recognize similar ligands, including SFAs, suggests that Tlr2 and
Tlr4 are major mediators of obesity-induced inflammation. In the present study, the more
pronounced effect of palmitate may result from a combination of signaling through both of
Our second major finding pertains to the importance of JNK activity to ROS
the exact mechanism is not yet known, NEFA were associated with the generation of ROS in
adipocytes (12). Lee and colleagues (43) recently demonstrated that a Tlr-JNK pathway
121
suggested that JNK mediates insulin resistance through generation of oxidative stress. Our
data show that palmitate, LPS, and ZymA all induce JNK phosphorylation (activation), iNOS
expression and ROS accumulation in adipocytes. Furthermore, the inhibitor used to disrupt
activation of JNK not only blocked or attenuated the ability of palmitate, LPS and ZymA to
stimulate phosphorylation of JNK, it also precluded the induction of ROS and the diminution
specificity. However, it should be noted that SP600125 is shown to have >20-fold selectivity
for JNK compared to a range of other kinases (45). Future experiments utilizing Tlr
knockdown models will be necessary to further validate the specificity of SP6 to JNK. The
confirm prior evidence (46) detailing the importance of ROS as determinants of insulin
component of the inflammatory response to palmitate, LPS, and ZymA, and presented initial
evidence that activation of JNK may be required for activation of AP-1 by palmitate and
ZymA, but not LPS. The fact that we showed a limited effect of JNK inhibition on LPS-
induced AP-1 activation may relate to distinct differences between Tlr4 and Tlr2 signaling
(47). For instance, LPS and other Tlr4 agonists are capable of signaling through both
MyD88-dependent and independent pathways, whereas Tlr2 ligands require the MyD88
122
study, activation of NFB was induced by palmitate and both Tlr agonists. Moreover, the
Tlr4 agonist, LPS, had the greatest increase in NFB activation (Fig. 3C), whereas the Tlr2
agonist, ZymA, had the greatest induction of AP-1 (Fig. 3D). Involvement of LPS in NFB
activation in adipocytes has been well documented (21,36). However, we have established
herein that the Tlr2 agonist, ZymA, not only activates NFB, but also induces AP-1 in
adipocytes. The reduced ability of JNK inhibitor to fully inhibit LPS activation of AP-1 also
The in vivo activation of AP1 and NFB in AT is likely a concerted process involving
transcription factors promotes lipolysis (41), leading to increased NEFA and activation of Tlr
signaling within adipocytes and neighboring cells. However, elucidating the contribution of
different AT-based cells to Tlr2 and Tlr4 mediated inflammation will require further
investigation.
palmitate, in adipocyte inflammation, oxidative stress, and insulin resistance. These data also
indicated that JNK activation is essential to ZymA agonist induced NFB and AP-1
activation. Based on these data, we believe that Tlr2, in addition to Tlr4, is an important
mediator of obesity-induced inflammation. Further insight into this relationship may provide
potential therapeutic strategies to inhibit Tlr agonists for alleviating obesity-associated health
complications.
123
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128
Treatment Effect P < 0.0001 P < 0.0001 P < 0.0001 P < 0.0001 P = 0.0009
a, b, c, d
Significant difference between LS means indicated by different letters (P<0.05)
129
a, b, c
Significant difference between LS means indicated by different letters (P<0.05)
130
Figure 8. Palmitate, ZymA, and LPS induced insulin resistance in 3T3-L1 adipocytes.
10-12 d post-differentiated 3T3-L1 adipocytes were treated with 0.5 mM sodium palmitate,
10 g/mL LPS, or 20 g/mL ZymA for 3, 6, 8, 12, 24, and 48 h. Insulin-stimulated 2-DG
uptake was then determined (n = 8, Treatment effect P<0.0001, Time effect P<0.0001,
Treatment*Diet effect P<0.0001). A non-insulin stimulated contol was included for
reference. Results are expressed as fold change versus insulin stimulated control and
represent LS means SEM. When Treatment effect was significant (P<0.05), post-hoc t-test
was used to determine significant difference between LS means as indicated by different
letters.
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Figure 9. Palmitate, ZymA, and LPS induced JNK phosphorylation and NFB and AP-1
nuclear translocation in 3T3-L1 adipocytes.
10-12 d post-differentiated 3T3-L1 adipocytes were treated with 0.5 mM sodium palmitate,
10 g/mL LPS, or 20 g/mL ZymA for 48 h (n= 4 unless noted otherwise). (A)
Representative western blot showing phosphorylation of JNK (top panel) and total JNK
levels (bottom panel). (B) Quantification of phosphorylated JNK by densitometry and
expressed as fold change versus control (Treatment effect P=0.0089). Representative EMSA
blot showing nuclear protein binding to labeled (C) NFB and (D) AP-1 consensus
sequences.
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Figure 10. JNK inhibitor, SP600125, attenuated palmitate, ZymA, and LPS induced JNK
phosphorylation and inflammation in 3T3-L1 adipocytes.
10-12 d post-differentiated 3T3-L1 adipocytes were treated with 0.5 mM sodium palmitate,
10 g/mL LPS, or 20 g/mL ZymA for 48 h (n=6-8 unless noted otherwise). (A)
Representative western blot showing phosphorylation of JNK (top panel) and total JNK
levels (bottom panel). (B) Quantification of phosphorylated JNK by densitometry (Treatment
effect P<0.0001). (C) IL-6 gene expression (Treatment effect P<0.0001). (D) MCP-1 gene
expression (Treatment effect P<0.0001). (E) Tlr2 gene expression (Treatment effect
P<0.0001). (F) Tlr4 gene expression (Treatment effect P=0.0002). All displayed were
expressed as fold change versus control and represent LS means SEM. When Treatment
effect was significant (P<0.05), post-hoc t-test was used to determine significant difference
between LS means as indicated by different letters.
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Figure 11. JNK inhibitor, SP600125, attenuated palmitate, ZymA, and LPS induced NFB
and AP-1 nuclear translocation, oxidative stress, and insulin resistance in 3T3-L1
adipocytes.
10-12 d post-differentiated 3T3-L1 adipocytes were treated with 0.5 mM sodium palmitate,
10 g/mL LPS, or 20 g/mL ZymA for 48 h (n= 6-8 unless noted otherwise). Representative
EMSA blot showing nuclear protein binding to labeled (A) NFB and (B) AP-1 consensus
sequences. (C) iNOS gene expression (Treatment effect P<0.0001). (D) DCF fluorescence
(Treatment effect P<0.0001). (E) Insulin-stimulated 2-DG uptake (Treatment effect
P<0.0001). All displayed values represent LS means SEM for Treatment effect. When
Treatment effect was significant (P<0.05), post-hoc t-test was used to determine significant
difference between LS means as indicated by different letters.
134
Abstract
acid (SFA)-induced inflammation and insulin resistance. However, less is known about toll-
like receptor-2 (Tlr2), which also recognizes SFA. To investigate the involvement of this
receptor in diet-induced obesity, we utilized male Tlr2-/- mice and C57BL6J controls. We
also compared 10Scn mice lacking functional Tlr4 and their respective control 10J. Mice
were fed a low fat (low-fat control (LFC)), high unsaturated fat (high-fat control (HFC)), or
high saturated fat + palmitate (HFP) diet ad libitum for 16 weeks. Tlr2-/- mice were protected
from DIO. However, unlike Tlr4 deficient 10ScN mice, there were no significant changes in
body weight, energy intake, or lipid profile in Tlr2-/- mice fed any of the three diets as
endotoxin, and monocyte chemotactic protein-1 (MCP-1) were lower in 10ScN mice fed
HFP diet, whereas only MCP-1 levels were reduced in high fat fed Tlr2-/- mice. Transcript
abundance of adipose markers of inflammation and oxidative stress, including tumor necrosis
factor-, interleukin-6, MCP-1, F4/80, and nitric oxide synthase-2, were attenuated by Tlr2
and Tlr4 deficiency. Furthermore, these effects were more striking in HFP-fed mice. Blood
glucose and serum insulin were lower in 10ScN mice, but not in Tlr2-/- mice. However,
stromal vascular (SV) cells isolated from AT of Tlr2-/- had a marked increase in insulin
sensitivity. These data indicated that Tlr2, as well as Tlr4, play a significant role in the
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a HFP diet.
Introduction
risk for diabetes mellitus (DM) (1-3). Obese individuals exhibit activation of several distinct
inflammatory pathways, including nuclear factor kappa B (NFB) and c-jun N-terminal
kinase (JNK) (1,2), which are associated with macrophage infiltration in AT and stimulation
weight patients. The strong association between obesity, inflammation, and insulin
resistance suggests that adipose tissue (AT) plays a prominent role in their development.
of the obese is not fully elucidated, but may involve signaling through specific Tlrs. Shi and
colleagues (4) reported attenuation of fatty acid (FA)-induced NFB activation and insulin
resistance in AT of Tlr4 null mice. Additional studies (5-8) have confirmed that Tlr4
(DIO). We previously demonstrated that 10ScN mice, which have a 74 kb deletion from
chromosome 4 that precludes expression and production of Tlr4, were specifically protected
from saturated fatty acid (SFA)-induced adiposity and AT-specific inflammation (9).
However, Tlr-4 deficiency did not fully attenuate high fat diet-induced inflammation in AT.
These data suggested that pathways independent of Tlr4 may also mediate obesity-induced
and diabetic individuals (10,11). Since SFA are reported to contribute to activation of Tlr2, it
is a likely mediator of obesity-induced inflammation (12). Thus, the goal of this study was to
test the hypothesis that the absence of Tlr2 expression attenuates systemic inflammation,
Animals and animal care. Male and female B6.129-Tlr2tm1Kir/J (Cat No. 004650;
Tlr2-/-) mice and C57BL/10ScN mice (Cat No. 003752; 10ScN) derived from the C57BL/6J
and C57BL/10J sublines, respectively, were obtained from Jackson Laboratories (Bar
Harbor, ME), along with corresponding control male and female C57BL/6J (Cat No. 000664)
and C57BL/10J (Cat No. 000665) mice. Breeding colonies were established for each
genotype. Aberrant Tlr2 and Tlr4 expression was confirmed by real time PCR. At 6-8 weeks
of age, male offspring were housed individually in a room with an automatically controlled
12-hour light:dark cycle. Mice were acclimated to the cages and provided unlimited access to
Animals from each genotype were randomly assigned to one of three experimental
diets (n = 15/genotype/diet): 1) low fat control (LFC), high fat control (HFC), or high fat
palmitate (HFP) for 16 weeks. Both high fat diets were semi-purified, powdered diets based
obesity by providing 60% of calories from lipids (Table 6). The high fat diets were identical
except the source of fat used was either soybean oil in the HFC (Harlan Teklad) or a mixture
of lard (Harlan Teklad) and purified palmitate (Nu-Check Prep, Inc.) in the HFP diet. The
LFC diet contained 12% of calories from lipid. Food intake was measured daily and used to
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calculate total energy intake. Mice were fasted overnight (6-8 h) and euthanized by CO2
asphyxiation for blood and tissue collections. Blood was collected by heart puncture, placed
on ice, and serum was collected by centrifugation and frozen at -80oC until analyzed.
Animals were weighed and abdominal fat pads were removed and weighed. Extracted tissues
and carcasses were immediately frozen in liquid nitrogen and stored at -80C prior to RNA
extraction and immuno-histochemical analysis. All experimental protocols for animal care
and use were approved by the Institutional Animal Care and Use Committee at Iowa State
Technology Systems, Inc.). Serum insulin (Linco), C-reactive protein (CRP) (Immunology
Consultants Laboratory, Inc.), and MCP-1 (R&D Systems) were measured using commercial
assay kits. Non-esterified fatty acids (NEFA) were measured with NEFA-C kit (Wako Pure
Chemical Industries, Inc.). Serum endotoxin determinations were performed using a kit
based upon a Limulus amaebocyte extract (LAL kit; Hycult Biotechnology). Samples were
diluted 1:40. Internal control of recovery calculation was included in the assessment.
Quantitative real-time PCR. Total RNA was extracted and cDNA synthesized as
described previously (9). Primer sequences are listed in Table 7. The abundance of each gene
product was calculated by regressing against the standard curve generated in the same
reaction with their respective plasmid. All genes of interest were normalized to beta actin and
Serum fatty acid profile. Lipid from serum samples were extracted by the method of
Lepage and Roy (15) with minor modifications as described previously (9).
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Stromal vascular cell (SVC) isolation and culture. Male Tlr2-/- or 6J mice were
euthanized by CO2 asphyxiation. Epidydimal fat pads were removed, trimmed, and diced
before adding to sterile digestion cocktail (1X Krebs Ringer buffer (KRB) and 200 U/ml
collagenase) for 1 h in 37C shaking water bath. Inter-phase liquid, absent of floating
adipocytes and debris, was centrifuged at 800 x g for 4 min. The resulting pellet was
resuspended in red blood cell lysis buffer (pH 7.2; 155 mM HH4Cl, 5.7 mM K2HPO4, and 0.1
mM EDTA) for 5-6 min and then diluted with 1X PBS and centrifuged for 800 x g for 15
min. Following a second PBS wash, the pellet was resuspended in low glucose DMEM
containing 10% FBS and 5% streptomycin/penicillin mixture. Growth media was changed
after 4 h and then again at 24 h, after which media was replaced every 48 h. Cells were split
twice before being frozen in media containing 15% DMSO. These stocks were subsequently
media containing 1.5 mM IBMX, 1.0 M dexamethasone, 1.7 M insulin, and 0.4 mM
biotin) was added for 48 h. Following an additional 48 h with differentiation cocktail, cells
of SVC exhibited adipocyte phenotype (e.g., lipid accumulation). Cells were then treated
with 0.5 mM sodium palmitate, 10 g/mL LPS from Escherichia coli 055:B5, or 20 g/mL
ZymA from Saccharomyces cerevisiae for 48 h and used thereafter for analysis of glucose
uptake.
2-Deoxyglucose (DG) Uptake. SV cells were treated as described and glucose uptake
Statistical analyses. Data were tested for normality and analyzed using the mixed-
model analysis with diet and genotype considered fixed effects, and the time dietary
main effects are indicated as Diet*Genotype effects. All indicated P-values were two-tailed
and Bonferroni corrected. Differences were considered significant at P < 0.05 and a tendency
at P < 0.10. Values are presented as least squares (LS) means SEM.
Results
Tlr2-/- and 10ScN mice are protected from diet-induced obesity independent of caloric
intake. Unlike 10ScN mice, Tlr2-/- mice did not exhibit a reduction in body weight (Fig.
12A) or weight gain (Fig. 12B) compared to 6J controls (Genotype, P= 0.68). In contrast,
10ScN mice had on average 2.9 g lower body weight relative to 10J mice (Genotype,
P=0.0018). This relationship was especially evident in HFP fed 10ScN mice (Diet*Genotype,
P=0.0076) and is consistent with our previous findings (9). To further evaluate the level of
adiposity, we measured epidydimal fat pad weight (Fig. 12C) and observed that both Tlr2-/-
and 10ScN mice had approximately 20% and 30% lower fat pad weight compared to their
controls, respectively (Genotype, P<0.0001). Epidydimal fat pad weight was higher in high
fat diet fed mice (Diet Effect, P=0.032) relative to LFC fed mice. These effects on fat pad
weight were independent of energy intake as there were no intake differences between Tlr2-/-
and 6J mice (Fig. 12D, Genotype, P=0.58). However, 10ScN mice actually exhibited greater
energy intake than 10J mice (Genotype, P=0.0097), which was especially evident in HFC fed
Tlr4 deficiency, but not Tlr2 deficiency, improved markers of insulin sensitivity,
particularly in mice fed a high saturated fat diet. In the present study, we measured fasting
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blood glucose (Fig. 13A) and serum insulin (Fig. 13B) to determine if Tlr2-/- mice might also
exhibit modification of insulin sensitivity. We have shown that blood glucose concentration
was over 10% lower in 10ScN mice versus 10J controls (Genotype, P=0.019). However, we
observed no significant difference in blood glucose or serum insulin between Tlr2-/- and 6J
controls (Genotype, P=0.12 and P=0.66, respectively). Dietary treatment also had no effect
on blood glucose regardless of genotype (Diet, P=0.93). In contrast, serum insulin was
approximately 30% higher in mice fed HFP compared to HFC (Diet, P=0.035) suggesting
that a diet rich in SFA is more insulinemic compared to a diet rich in unsaturated FA. One
caveat to this was that 10ScN mice had a tendency toward lower insulin concentration when
13C), endotoxin (Fig. 13D), and MCP-1 (Fig. 13E). Circulating CRP concentration was
greater in mice fed a saturated versus unsaturated fat diet (P=0.031), but this effect was
blunted in Tlr4 deficient mice fed HFP diet (Diet*Genotype, P=0.0034). Furthermore, Tlr4
deficient mice showed a nearly 30% reduction in serum endotoxin regardless of diet
endotoxin between Tlr2-/- and 6J mice (P=0.46 and P=0.79, respectively). Serum
10ScN mice versus 10J controls (Genotype, P=0.0035). The reduction in MCP-1
concentration was more evident in Tlr4 deficient mice fed either high fat diet
(Diet*Genotype, P=0.035 (HFC) and P=0.05 (HFP), respectively). Tlr2-/- mice were also
protected from the HFP diet-induced MCP-1 increase (Diet*Genotype, P=0.0038), but not
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from the HFC diet (Diet*Genotype, P=0.58). Therefore, it appeared that Tlr2, in addition to
Tlr4, was involved in the stimulation of the SFA diet-induced serum MCP-1.
response to high fat diets. We measured both the concentration and composition of serum
shown that mice fed either high fat diet had a near 20% higher serum total cholesterol (Table
8, Diet, P=0.0075 (HFC) and P<0.0001 (HFP), respectively), whereas serum TAG (Table 8)
was elevated in LFC fed mice regardless of genotype (Diet, P< 0.0001). Furthermore, 10ScN
mice exhibited approximately 20% greater serum TAG compared to 10J controls (Genotype,
P=0.0049), which was more apparent in 10ScN mice fed LFC diet (Diet*Genotype,
P<0.0001). Similarly, circulating NEFA (Table 8) were higher in 10ScN mice fed the LFC
diet (Diet*Genotype, P<0.01). There were no observed differences in serum TAG or NEFA
between Tlr2-/- mice and 6J controls (Genotype P=0.97 and P=0.85, respectively).
We next analyzed the composition of serum FA (Table 9). Total SFA, palmitate, and
stearate content were all 21%, 14%, and 35% higher in serum of mice fed HFP and LFC
compared to HFC diet, respectively (Diet, P<0.0001). In contrast, total omega-6 and omega-
3 FA content were approximately 18% and 25% greater in serum of mice fed HFC than HFP
diet (Diet, P<0.0001), which corresponds with higher serum linoleate, -linolenate, and DHA
(Diet, P<0.0001). Polyunsaturated FA (PUFA) content was also different between LFC and
HFP fed mice. Total omega-3 FA was greater in mice fed LFC (Diet, P<0.0001), whereas
total omega-6 FA was greater in mice fed HFP (Diet, P=0.0029), which is consistent with the
observed DHA (Diet, P<0.0001) and -linoleate concentrations (Diet, P<0.01), respectively.
Genotype had a minimal impact on FA composition. However, Tlr2-/- mice exhibited higher
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circulating stearate (Genotype, P=0.0003) with a trend toward an increase in total SFA
linolenate (Genotype, P=0.0067) and linoleate (Genotype, P=0.0960) versus 10J controls.
These data indicated that the circulating lipid profile was more closely related to dietary lipid
content than genotype, thus indicating that the anti-inflammatory effects of Tlr2 or Tlr4
saturated and unsaturated fat diets, and was partly ameliorated by Tlr2 and Tlr4 deficiency.
We first confirmed that Tlr4 (Fig. 14A) and Tlr2 (Fig. 14B) gene expression were deficient
of Tlr4 was 2-fold greater in mice fed either high fat diet (Diet, P=0.0068 (HFP) and P=0.020
(HFC), respectively). SFA fed mice also exhibited a 3-fold increase in Tlr2 transcript levels
inflammation, we measured transcript abundance of TNF (Fig. 14C), IL-6 (Fig. 14D), and
MCP-1 (Fig. 14E) in AT of Tlr2-/-, 10ScN, and the respective control mice. Tlr2-/- mice had
2-fold lower TNF and MCP-1 transcript levels in AT compared to 6J mice (Genotype,
P<0.0001 and P=0.0087, respectively). Similarly, TNF transcript abundance was 2-fold
lower in 10ScN mice versus 10J controls (Genotype, P=0.0269). Tlr2-/- mice were
specifically protected from HFP diet-induced TNF, IL-6, and MCP-1 expression in AT
IL-6 and MCP-1 transcript abundance by 2-fold in AT of HFP fed mice (Diet*Genotype,
P=0.045 and P=0.046). These findings indicated that the ability of a high saturated fat diet to
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We have shown that transcript abundance of macrophage marker, F4/80, was over 2-
fold lower in Tlr2-/- and 10ScN mice relative to respective controls (Genotype, P=0.025 and
abundance in HFP fed mice F4/80 (Diet*Genotype, P=0.0014 and P=0.0045, respectively).
In the present study, nitric oxide synthase-2 (NOS2) transcript abundance was 5.2-
and 5.9-fold lower in both Tlr2-/- and 10ScN mice compared to 6J and 10J controls
(Genotype, P<0.0001). This effect was more prominent with either high fat diet (Diet,
P<0.0001), but the highest level of NOS2 transcript was in HFP fed mice (Diet, P=0.030).
The ability of Tlr2 or Tlr4 deficiency to attenuate HFC-induced NOS2 was similar
(Diet*Genotype, P<0.0001). However, only 10ScN mice exhibited a protective effect against
the HFP diet (Diet*Genotype, P<0.0001). Overall, these results indicated that Tlr2 and Tlr4
Tlr2 deficient SV cells were protected from FFA-induced inflammation and insulin
resistance. We observed a limited effect of Tlr2 deficiency on blood glucose and serum
insulin. These findings may reflect an increased susceptibility to DIO on the 6J background,
which was clearly demonstrated by the greater body weight, adiposity, and energy intake in
mice on 6J background (Tlr2-/- and 6J), relative to 10J background (10ScN and 10J)
improved at the cellular level. To do this we isolated and cultured primary SV cells from AT
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of Tlr2-/- and 6J mice. Confluent cells were then treated with adipocyte differentiation media
(~40% of cells exhibited lipid accumulation) and used for determination of 2-DG uptake.
cells, regardless of treatment (Fig. 15, Genotype, P<0.0001). Furthermore, Tlr2 deficient SV
cells treated with palmitate, LPS, or ZymA for 48 hr had improved insulin sensitivity
improved insulin sensitivity in SV cells isolated from AT, but failed to completely block
Discussion
Obesity is associated with chronic low grade inflammation, which contributes to the
development of insulin resistance (17). The molecular mechanisms involved are not fully
elucidated but may involve activation of Tlr2 and Tlr4 signaling pathways in AT. Several
recent studies (4,6,7,20) have identified a role of Tlr4 in obesity and FA-induced
inflammation. Herein, we demonstrate that Tlr2-/- mice were protected from DIO without
significant changes in energy intake or lipid profile. These mice also exhibited a dramatic
mice had a marked increase in insulin sensitivity. These data indicated that Tlr2 is involved
Diets high in SFA are associated with increased inflammation (21). We have
previously reported that Tlr4 deficiency specifically protected against a high SFA diet-
induced adiposity and inflammation (9). In this study, we confirmed this effect in 10ScN
mice, but also demonstrated that absence of functional Tlr2 blunted the high SFA diet-
induced inflammation. Moreover, the attenuated inflammatory response in Tlr2-/- and 10ScN
mice suggests that the absence of these receptors protects against the elevated serum SFA
observed in HFP-fed control mice. The ability of SFA to induce inflammation through Tlr4 is
well documented in vitro (5,22). However, only a few studies (12,23) have implicated Tlr2 as
a potential target for SFA, including Senn (23), who reported that Tlr2 siRNA reversed
the idea that Tlr2 is an endogenous target for SFA, and thus promotes obesity-induced
inflammation.
signaling in AT often precedes the onset of systemic insulin resistance (24). We previously
reported that Tlr4 deficient mice exhibited lower macrophage infiltration in AT (9). In the
present study, we observed that transcript abundance of the macrophage specific marker,
F4/80 was markedly reduced in Tlr2 and Tlr4 deficient mice. This reduction was more
apparent in HFP-fed mice, and corresponded with lower expression levels of multiple
proinflammatory genes (e.g. TNF, IL-6, and MCP-1). Additionally, the high fat diet-
induced NOS2 transcript was reduced in Tlr2-/- and 10ScN mice, which supports our
previous findings that Tlr2 and Tlr4 ligands induce ROS in 3T3-L1 adipocytes (16). Thus,
146
Tlr2 and Tlr4 deficiency protected against AT inflammation and oxidative stress, especially
insulin resistance. Alternatively, Caricilli et al. (25) showed that injection of Tlr2 antisense
Furthermore, inhibition of Tlr2 also improved insulin sensitivity in AT and muscle of DIO
mice. Our data showed that only 10ScN mice exhibited improved serum markers of insulin
insulin sensitivity in primary SV cells from Tlr2-/- mice. Although we observed a marked
improvement in insulin sensitivity in the Tlr2-/- mice, the absence of Tlr2 failed to fully
protect against palmitate-induced insulin resistance. These findings indicate that Tlr2 does
resistance as is Tlr4. In summary, our study demonstrates a requisite role of Tlr2 and Tlr4 in
obesity-induced inflammation and insulin resistance. The absence of these receptors protects
against systemic and AT specific inflammation, especially in mice fed a high SFA diet. We
therefore assert that both Tlr2 and Tlr4 represent potential dietary and pharmaceutical targets
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150
Table 8. Lipid profiles in Tlr2 and Tlr4 deficient mice and wild type controls fed LFC, HFC,
or HFP diet for 16 weeks
Genotype Diet Serum Serum Serum
Cholesterol Triacylglyceride NEFA
________________________mmol/L______________________
10ScN LFC 5.00 0.59c 1.53 0.08a 2.48 0.29a
bc c
10ScN HFC 6.39 0.53 0.73 0.07 1.51 0.30b
bc bc
10ScN HFP 6.17 0.51 0.75 0.07 1.48 0.39b
Main Effects
Genotype P < 0.0001 P = 0.0302 P = 0.019
Diet P = 0.0001 P < 0.0001 P = 0.36
Interaction
Diet*Genotype P = 0.13 P < 0.0001 P = 0.052
a, b, c, d
Significant difference between LS means indicated by different letters (P<0.05)
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Table 9. Serum fatty acid profiles in Tlr2 and Tlr4 deficient mice and wild type controls fed
LFC, HFC, and HFP diet for 16 weeks
a, b, c, d
Significant difference between LS means indicated by different letters (P<0.05)
154
Figure 12. Tlr2-/- and 10ScN mice were protected against DIO.
All measures were taken after 16 weeks of LFC (white bars), HFC (black bars), or HFP (grey
bars) diet (n = 15 unless noted otherwise). (A) Body weight of 6J, Tlr2-/-, 10ScN, and 10J
mice (Diet effect P= 0.58, Genotype effect, P<0.0001, Diet*Genotype interaction P=0.0085).
(B) Total weight gain of 6J, Tlr2-/-, 10ScN, and 10J mice (Diet effect P= 0.58, Genotype
effect, P<0.0001, Diet*Genotype interaction P=0.0085). (C) Epididymal fat pad weight of
6J, Tlr2-/-, 10ScN, and 10J mice (Diet effect P=0.032, Genotype effect P<0.0001,
Diet*Genotype interaction P=0.040). (D) Total energy intake (kcal) of 6J, Tlr2-/-, 10ScN, and
10J mice (Diet effect P=0.82, Genotype effect P<0.0001, Diet*Genotype interaction
P=0.013). All displayed values were adjusted for initial body weight and represent LS means
SEM for Diet*Genotype interaction. When main effects (diet and genotype) or interaction
term were P<0.15, Bonferroni correction was used to determine significant difference
between means as indicated by different letters.
155
Figure 13. Absence of Tlr2 and Tlr4 expression differentially modified markers of systemic
insulin sensitivity and inflammation.
All measures were taken after 16 weeks of LFC (white bars), HFC (black bars), or HFP (grey
bars) diet. (A) Blood glucose from 6-8 h fasted 6J, Tlr2-/-, 10ScN, and 10J mice (n = 15, Diet
effect P= 0.81, Genotype effect, P=0.066, Diet*Genotype interaction P=0.65). (B) Serum
insulin (n = 5, Diet effect P=0.096, Genotype effect P=0.0002, Diet*Genotype interaction
P=0.15). (C) Serum CRP from 6J, Tlr2-/-, 10ScN, and 10J mice (n = 5, Diet effect P=0.095,
Genotype effect, P<0.0001, Diet*Genotype interaction P=0.066). (D) Serum endotoxin (n =
5, Diet effect P=0.57, Genotype effect P<0.0001, Diet*Genotype interaction P=0.34). E)
Serum MCP-1 (n = 5, Diet effect P=0.42, Genotype effect P=0.0002, Diet*Genotype
interaction P=0.090). All displayed values were adjusted for initial body weight and
represent LS means SEM for Diet*Genotype interaction. When main effects (diet and
genotype) or interaction term were P<0.15, Bonferroni correction was used to determine
significant difference between means as indicated by different letters.
156
Figure 14. Absence of Tlr2 and Tlr4 expression protected against SFA-induced inflammation
in AT.
All measures were taken after 16 weeks of LFC (white bars), HFC (black bars), or HFP (grey
bars) diet. (A) Tlr4 transcript abundance in AT of 6J, Tlr2-/-, 10ScN, and 10J mice (n = 6,
Diet effect P=0.014, Genotype effect, P<0.0001, Diet*Genotype interaction P=0.24). (B)
Tlr2 transcript abundance in AT of 6J, Tlr2-/-, 10ScN, and 10J mice (n = 6, Diet effect
P=0.0031, Genotype effect P<0.0001, Diet*Genotype interaction P=0.073). (C) TNF
transcript abundance in AT of 6J, Tlr2-/-, 10ScN, and 10J mice (n = 6, Diet effect P=0.66,
Genotype effect P<0.0001, Diet*Genotype interaction P=0.040). (D) IL-6 transcript
abundance in AT of 6J, Tlr2-/-, 10ScN, and 10J mice (n = 6, Diet effect P=0.34, Genotype
effect P=0.0005, Diet*Genotype interaction P=0.015). (E) MCP-1 transcript abundance in
AT of 6J, Tlr2-/-, 10ScN, and 10J mice (n = 6, Diet effect P=0.13, Genotype effect P<0.0001,
Diet*Genotype interaction P=0.14). (F) F4/80 transcript abundance in AT of 6J, Tlr2-/-,
10ScN, and 10J mice (n = 6, Diet effect P=0.19, Genotype effect P<0.0001, Diet*Genotype
interaction P=0.084). (G) NOS2 transcript abundance in AT of 6J, Tlr2-/-, 10ScN, and 10J
mice (n = 6, Diet effect P<0.0001, Genotype effect P<0.0001, Diet*Genotype interaction
P<0.0001). All displayed values were adjusted for initial body weight and beta actin
transcript level. Values represent LS means SEM for Diet*Genotype interaction. When
main effects (diet and genotype) or interaction term were P<0.15, Bonferroni correction was
used to determine significant difference between means as indicated by different letters.
157
Figure 15. SV cells isolated from Tlr2-/- exhibit improved insulin sensitivity, but were not
fully protected against FA-induced insulin resistance.
CHAPTER 6. SUMMARY
The rise in obesity corresponds to the development of DM and CVD (1,2). The
molecular mechanism linking obesity to these inflammatory diseases is not fully elucidated
from adipocytes, which signals neighboring or downstream cells (6). The ability of FFA,
innate immune receptors, Tlr2 and Tlr4. Several studies (7-9) have implicated the
various cell populations. However, little is known about the relationship of these receptors in
precludes expression of Tlr4, were protected against DIO. This effect was more prominent in
mice fed a high SFA diet. Moreover, these mice exhibited a specific reduction in AT
activation and subsequent gene expression. There was also a decrease in macrophage
accumulation in AT of mice fed the HFP diet. These data indicated that Tlr4 was involved in
high fat diet-induced adiposity and inflammation in AT. However, Tlr4 deficiency did not
fully attenuate TNF and IL-6 transcript abundance in AT of high saturated diet fed mice.
Corresponding to these findings was an increase in Tlr2 expression, which because of its
159
inflammation.
adipocytes to investigate the ability of the Tlr2 agonist, ZymA, to mediate inflammation and
insulin resistance. Our data showed that ZymA induced proinflammatory gene expression
similarly to palmitate and LPS treatment. Furthermore, the Tlr2 agonist stimulated NFB
nuclear translocation, although not to the same extent as the Tlr4 agonist, LPS. We also
demonstrated for the first time in adipocytes that ZymA caused nuclear translocation of the
inflammation was apparent. Along with the proinflammatory response observed with ZymA,
LPS, and palmitate treated cells was an increased accumulation of ROS, which closely
evaluated insulin-stimulated glucose uptake in adipocytes treated with SFA or Tlr agonist
Activation of the intracellular kinase, JNK, is associated with Tlr signaling (7,12)
thus, we investigated the ability of a highly specific JNK inhibitor to reduce inflammation
and oxidative stress, as well as improve insulin sensitivity. There was a marked attenuation
of insulin resistance in ZymA, LPS, and palmitate treated adipocytes, which corresponded to
a reduction in NFB and AP-1 activation, proinflammatory gene expression, and ROS
accumulation. From these data we concluded that Tlr2 is likely involved in obesity-induced
Additionally, it appeared that JNK activation was essential to the SFA and Tlr agonist
mediated effects.
Based on our studies with Tlr4 deficient mice and cultured adipocytes, we then
unsaturated) differentially modified inflammation and insulin resistance. Our data showed
that Tlr2-/- mice were protected from DIO. However, unlike Tlr4 deficient 10ScN mice, there
were no significant changes in body weight, energy intake, or lipid profile. These findings
indicated that Tlr2 deficiency significantly modified body composition, which may reflect
metabolic changes in energy metabolism. For instance, reduced adiposity in Tlr2-/- mice
endotoxin, and MCP-1 were lower in 10ScN mice fed a HFP diet, whereas only MCP-1 was
reduced in the Tlr2-/- mice fed a high fat diet. The disparity in these data may be due to
DIO, which in our studies was indicated by higher body weight and adiposity compared to
mice on 10J background. Therefore, the higher propensity toward obesity in Tlr2-/- mice may
background, both Tlr2 and Tlr4 deficiency improved AT markers of inflammation and
oxidative stress including TNF, IL-6, MCP-1, F4/80, and NOS2 transcript abundance.
Similar to our previous findings, the protective effect of Tlr deficiency was more apparent in
mice fed the high SFA diet. In Tlr2-/- mice, attenuation of AT inflammation did not correlate
with improved circulating glucose or insulin concentration. However, SV cells isolated from
161
AT of Tlr2-/- mice had a marked increase in insulin sensitivity, demonstrating the potential
The ability of both Tlr2 and Tlr4 to recognize FA and initiate proinflammatory
signaling in adipocytes, and other cell types, demonstrated the tremendous overlap of
fully elucidate the impact of Tlr2 and Tlr4 on adiposity and insulin resistance, energy
that Tlr2-/- and 10ScN mice had a greater protective effect when fed diet high SFA.
Therefore, future studies need to address the ability of various FA to induce an inflammatory
Overall, we have presented evidence that Tlr2 and Tlr4 were involved in obesity-
cellular level (i.e., adipocytes), which in turn reduced systemic inflammation and insulin
resistance. The underlying mechanism involved recognition of specific FA by Tlr2 and Tlr4,
and more specifically interaction with SFA. Based on these studies, Tlr2 and Tlr4 may
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