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The Effects of Saturated Fatty Acid Palmitate on Neuropeptide Gene
Expression, Signal Transduction, and Insulin Signaling in an Immortalized
Hypothalamic Neuronal Cell Model, mHypoA-NPY/GFP
By
Brian Wong
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Department of Physiology
University of Toronto
© Copyright by Brian Wong 2015
ii
The Effects of Saturated Fatty Acid Palmitate on Neuropeptide Gene
Expression, Signal Transduction, and Insulin Signaling in an Immortalized
Hypothalamic Neuronal Cell Model, mHypoA-NPY/GFP
Brian Wong
Master of Science
Department of Physiology
University of Toronto
2015
Abstract
Recent evidence suggests a role for hypothalamic insulin resistance in obesity
pathogenesis, and that obesity-associated hypothalamic inflammation underlies this
resistance. However, few studies have examined the direct effects of saturated fatty acids on
specific hypothalamic neurons. Therefore, an immortalized hypothalamic neuronal cell
model expressing NPY and AgRP was used to determine the effects of palmitate on
neuropeptide gene expression, signal transduction events and insulin signaling. In the
mHypoA-NPY/GFP neuronal cell model, palmitate was found to upregulate the expression
of NF-κB and IκBα within 4 hours of treatment, and upregulate expression of AgRP after 24
hours of treatment. Regulation of AgRP gene expression appeared to be palmitate
metabolism-dependent. Palmitate also induced p38 MAPK phosphorylation, and prolonged
palmitate pre-treatment decreased levels of phosphorylated Akt following insulin re-
challenge. This is the first evidence of palmitate-mediated changes in AgRP gene expression
and its signaling through p38 MAPK in a representative NPY/AgRP neuronal cell model.
iii
Acknowledgements
First and foremost, I owe my deepest gratitude to Dr. Denise Belsham. I am grateful
to have had you as my mentor and supervisor. The last two years in the laboratory have been
an incredible learning experience. Your continual guidance, support, and encouragement
have enabled me to grow as a person, and have prepared me for the road ahead. Thank you
Denise.
I would also like to thank my committee members, Dr. Michael Wheeler, Dr. Amira
Klip, and Dr. Adria Giacca. Your guidance, mentorship, and valuable insight have been
crucial to the completion of this degree.
I would like to thank my fellow lab mates, who made the laboratory an enjoyable
place to work in. Whether it was generating discussion at lab meetings or simply lending a
helping hand with a new experiment, you have all contributed to this experience.
Finally, I would like to thank my family for their unwavering love and
encouragement. The sacrifices you have made and the many opportunities you have provided
me with do not go unnoticed. I would not be where I am today without you.
iv
Table of Contents
Acknowledgements .......................................................................................................... iii
Table of Contents ............................................................................................................. iv
List of Tables and Figures ............................................................................................. viii
List of Abbreviations ....................................................................................................... ix
Chapter 1 Introduction
1.1 Preface................................................................................................................2
1.2 The Central Melanocortin System and Energy Homeostasis
1.2.1 Adiposity Negative Feedback ..........................................................2
1.2.2 Insulin as an Adiposity Signal .........................................................3
1.2.3 The Hypothalamus ...........................................................................4
1.2.4 The Role of Neuropeptides in the Brain ..........................................6
1.2.5 The Melanocortin System ................................................................6
1.2.6 Neuropeptide Y and Agouti-Related Peptide ..................................7
1.3 Glucose Sensing in Hypothalamic Neurons
1.3.1 Glucose Entry into the Brain and the Discovery
of Glucose Sensing Neurons ............................................................8
1.3.2 Glucose-Excited Neurons ..............................................................10
1.3.3 Glucose-Inhibited Neurons ...........................................................11
1.4 Fatty Acid Sensing in Hypothalamic Neurons
1.4.1 Fatty Acids and Metabolic Physiology ..........................................12
1.4.2 Blood-Brain Barrier Permeability, Fatty Acid Uptake and
Metabolic Fates ..............................................................................13
v
1.4.3 Fatty Acids as Signaling Molecules in the Hypothalamus ............13
1.5 Obesity and Hypothalamic Inflammation
1.5.1 High-Fat Feeding is Associated with Inflammation ......................15
1.5.2 Evidence that Hypothalamic Inflammation Contributes to
HFD-Induced Obesity ....................................................................16
1.5.3 Palmitate as a Mediator of Hypothalamic Inflammation ...............17
1.6 Obesity and Hypothalamic Insulin Resistance
1.6.1 Obesity, Diabetes and Insulin Resistance ......................................19
1.6.2 Hyperinsulinemia and the Development of Insulin Resistance .....20
1.6.3 High Fat Feeding and Neuronal Insulin Resistance .......................20
1.6.4 FFA Metabolism and Insulin Resistance .......................................21
1.7 Cell Model
1.7.1 The Need for Cell Lines .................................................................24
1.7.2 Adult Hypothalamic Cell Lines (mHypoA-xx) .............................25
1.7.3 mHypoA-NPY/GFP Cell Line .......................................................26
1.8 Hypothesis and Aims .......................................................................................26
Chapter 2 Materials and Methods
2.1 Cell Culture and Reagents ...............................................................................32
2.2 Palmitate Preparation .......................................................................................32
2.3 TNF-α Preparation ...........................................................................................32
2.4 Insulin Preparation ...........................................................................................33
2.5 Quantitative RT-PCR .......................................................................................33
2.6 Western Blot Analysis .....................................................................................34
vi
2.7 Statistical Analysis ...........................................................................................35
Chapter 3 Results
3.1 Palmitate elicits an inflammatory response and upregulates
Agrp gene expression in mHypoA-NPY/GFP neurons ...................................37
3.2 TNF-α, a pro-inflammatory surrogate of palmitate, also
upregulates AgRP gene expression in mHypoA-NPY/GFP neurons ...............38
3.3 Palmitate-mediated regulation of AgRP gene expression
is metabolism-dependent .................................................................................40
3.4 Palmitate triggers the phosphorylation of p38 MAPK in
mHypoA-NPY/GFP neurons ...........................................................................43
3.5 Palmitate pre-treatment dampens the mHypoA-NPY/GFP
neurons’ response to insulin.............................................................................43
Chapter 4 Discussion
4.1 General Discussion ..........................................................................................48
4.2 Plasma Non-Esterified Fatty Acids and the Determination
of Palmitic Acid Concentrations in the Brain ..................................................49
4.3 Transcriptional Effects of Palmitate on mHypoA-NPY/GFP Neurons ...........52
4.4 Transcriptional Effects of TNF-α on mHypoA-NPY/GFP Neurons ...............55
4.5 Palmitate Metabolism and Signaling Dynamics in
mHypoA-NPY/GFP neurons ...........................................................................56
4.6 Palmitate Impairs Insulin Signaling in mHypoA-NPY/GFP Neurons ............58
4.7 Limitations .......................................................................................................60
4.8 Future Directions .............................................................................................63
vii
4.9 Conclusion .......................................................................................................64
References .........................................................................................................................66
viii
List of Tables and Figures
Table 1.1 Characterization of the mHypoA-NPY/GFP cell line .......................................30
Fig. 1.1 Schematic illustrating the PI3K-Akt pathway ........................................................5
Fig. 1.2 NPY/AgRP and POMC neurons are directly regulated by insulin .........................9
Fig. 1.3 Metabolic fates of palmitate upon entering the cell .............................................14
Fig. 1.4 Activation of the IKKβ/NF-κB pathway leads to inflammation
and impaired insulin signaling .............................................................................18
Fig. 1.5 Mechanisms of palmitate-mediated inhibition of insulin signaling .....................22
Fig. 1.6 Generation of the mHypoA-NPY/GFP cell line ...................................................27
Fig. 3.1 Saturated fatty acid palmitate upregulates pro-inflammatory and Agrp
gene expression ......................................................................................................39
Fig. 3.2 TNF-α upregulates pro-inflammatory and Agrp gene expression ........................41
Fig. 3.3 Methyl palmitate does not regulate Agrp gene expression ...................................42
Fig. 3.4 Palmitate induces phosphorylation of p38 MAPK ...............................................44
Fig.3.5 Prolonged palmitate or insulin exposure dampens the
insulin-mediated increase in phospho-Akt .............................................................46
ix
Abbreviations
AgRP agouti-related peptide
ARC arcuate nucleus
β-oxidation beta oxidation
BBB blood-brain barrier
cDNA complementary deoxyribonucleic acid
CNS central nervous system
CNTF ciliary neurotrophic factor
CPT-1 carnitine palmitoyltransferase-1
CREB cAMP response element binding protein
DAG diacylglycerol
DIO diet-induced obesity
DMEM Dulbecco’s modified eagle medium
DMN dorsomedial nucleus
DNA deoxyribonucleic acid
eIF2 eukaryotic initiation factor 2
ELK-1 ETS domain-containing protein-1
ER endoplasmic reticulum
ERK extracellular-related kinase
FABP fatty acid binding protein
FATP fatty acid transport protein
FBS fetal bovine serum
FFA free fatty acid
x
FOXO1 forkhead box protein 01
GLUT4 glucose transporter type 4
GPAT glycerol-3 phosphate acyltransferase
HFD high-fat diet
ICC immunocytochemistry
ICV intracerebroventricular
IκBα inhibitor of nuclear factor kappa B alpha
IKK-β inhibitor of IkappaB kinase beta
IL-1β interleukin-1 beta
IL-6 interleukin-6
IR insulin receptor
IRS insulin receptor substrate
JNK c-Jun N-terminal kinase
LHA lateral hypothalamic area
LPL lipoprotein lipase
MAPK mitogen-activated protein kinase
MC3/4R melanocortin 3/4 receptor
MPO medial preoptic area
mRNA messenger ribonucleic acid
miRNA microRNA
α-MSH alpha-melanocyte stimulating hormone
NEFA non-esterified fatty acid
NF-κB nuclear factor kappa B
xi
NPY neuropeptide Y
NTS nucleus of the solitary tract
PBS phosphate buffer saline
PCR polymerase chain reaction
PFA perifornical area
PI3K phosphatidylinositol 3-kinase
PKB protein kinase B
PKC protein kinase C
POMC proopiomelanocortin
PTP1B protein tyrosine phosphatase 1 B
PVN paraventricular nucleus
qRT-PCR quantitative reverse transcriptase polymerase chain reaction
RNA ribonucleic acid
siRNA small interfering RNA
SPT serine palmitoyltransferase
STAT signal transducer and activator of transcription
SV40 simian virus 40
T-Ag T-antigen
TG triglyceride
TAG triacylglycerol
TLR toll-like receptor
TNF-α tumor necrosis factor-alpha
T2DM type 2 diabetes mellitus
1
Chapter 1
Introduction
2
Introduction
1.1 Preface
Most overweight or obese individuals develop hyperlipidemia, low-grade
inflammation, and insulin resistance often leading to type 2 diabetes mellitus (T2DM). While
obesity and T2DM may both originate from a primary hypothalamic disease, little is known
about how specific neurons within the hypothalamus sense and respond to nutrient
(particularly fat) excess. The Belsham laboratory has generated several immortalized,
hypothalamic neuronal cell lines from primary fetal and adult hypothalamic neuronal cell
cultures, which have already provided insight into the direct control of neuropeptide
synthesis by nutrients at a mechanistic level not practical in the whole brain. The purpose of
this thesis was to evaluate the effects of palmitate (the most abundant non-esterified saturated
fatty acid) on neuropeptide gene expression, signal transduction events and insulin signaling
in an immortalized, hypothalamic neuronal cell model representative of the NPY/AgRP
neuron. Using the mHypoA-NPY/GFP cell line, I provide evidence of palmitate-mediated
changes in AgRP gene expression and the dependency of such changes on palmitate
metabolism. In addition, these studies begin to elucidate palmitate-mediated signal
transduction events and provide further evidence of palmitate’s ability to impair insulin
signaling in distinct hypothalamic neurons. Taken together, these studies have direct
relevance to the molecular mechanisms involved in the overall development of complex
metabolic disorders, such as obesity.
1.2 The Central Melanocortin System and Energy Homeostasis
1.2.1 Adiposity Negative Feedback
3
Despite daily variations in energy intake, the body fuel stored in adipose tissue
remains relatively constant over time (1). This observation suggests that short-term
differences in energy balance (the difference between energy consumed and energy
expended) may be offset in the long term by a mechanism that maintains overall energy
homeostasis. Indeed, changes in body fat content through dieting (2), behavior modification
(3) or experimental over-feeding (4) have been shown to induce compensatory responses that
restore adiposity to homeostatic levels.
To explain this phenomenon, Kennedy proposed that inhibitory signals were
generated in proportion to body fat stores and acted in the brain to reduce food intake (5).
Weight loss reduced the plasma levels of these inhibitory signals, causing food intake to
increase until body fat stores returned to normal levels (6).
1.2.2 Insulin as an Adiposity Signal
Insulin, a peptide hormone produced by the pancreatic β-cells, was the first hormonal
signal implicated in the central nervous system control of energy homeostasis (7). It provides
information regarding the amount of body fat stored and causes a long-term catabolic
response, decreasing food intake and increasing energy expenditure (8). Insulin is secreted
acutely in response to increases in blood glucose (i.e. after consumption of a meal) and its
levels are directly correlated to the extent of body adiposity (9). As in peripheral tissues,
insulin binds to its cognate receptor in the CNS. The receptor belongs to the family of
tyrosine kinase receptors, and binding of insulin to its receptor triggers an intracellular
signaling cascade (10).
4
Binding of insulin leads to rapid autophosphorylation of its receptor, followed by
tyrosine phosphorylation and recruitment of insulin receptor substrate (IRS) proteins. This
leads to activation of downstream pathways such as the phosphatidylinositol 3 kinase (PI3K)
and the mitogen-activated protein kinase (MAPK) cascades (11). Activation of PI3K results
in activation of protein kinase B/Akt and subsequent phosphorylation of the transcription
factor FOXO, which is a critical downstream regulator of energy homeostasis in the CNS
(Figure 1.1) (12).
The hypothalamus contains the highest concentration of insulin receptors (IR) in the
central nervous system. However, IRs are also expressed in the olfactory bulb, cerebral
cortex, cerebellum and hippocampus (13, 14). Neurons within the hypothalamus are capable
of sensing circulating insulin because of their location near the third ventricle, where insulin
can enter via a saturable transporter across the blood-brain barrier (15).
1.2.3 The Hypothalamus
The hypothalamus is a key brain region controlling energy homeostasis. Histological
techniques reveal nuclei as clusters of neurons within the hypothalamus that have distinct
neuronal phenotypes. These neurons express a specific complement of neuropeptides,
neurotransmitters and receptors. Classical lesion studies have shown that some of these
hypothalamic nuclei act as discrete “feeding” and “satiety” centres (16). Lesions of the
ventromedial, paraventricular or dorsal medial hypothalamus lead to hyperphagia, while
lesions of the lateral hypothalamus lead to hypophagia (17).
Besides regulating energy homeostasis, the hypothalamus is also the control centre
for many other endocrine processes. Physiological processes that are under hypothalamic
control include: stress, growth, temperature regulation, water balance, sexual behavior and
5
Insulin
Cell Membrane
IRS-1
PI3K
PIP3
PDK1
AKT FoxO1
FoxO1
Nucleus
Insulin Receptor
P
P
P
POMC AgRP
Fig. 1.1 Schematic illustrating the PI3K-Akt pathway.
Insulin binds to the insulin receptor, which is a receptor tyrosine kinase that autophosphorylates itself.
This allows IRS proteins to dock. IRS proteins are then activated, and can recruit PI3K which
phosphorylates PIP2 to PIP3. PIP3 acts as a docking site for PDK1 and AKT, allowing for the
phosphorylation of Akt by PDK1. Phosphorylation of Akt leads to its nuclear translocation where it
phosphorylates FoxO1 transcription factor. Phosphorylated FoxO1 can no longer repress POMC
expression and stimulate AgRP gene expression, which results in decreased feeding.
6
reproduction, and circadian rhythms. Situated below the thalamus, posterior to the optic
chiasm and surrounding the third ventricle, the hypothalamus has access to circulating factors
that cross the blood-brain barrier (BBB) via diffusion or saturable transport mechanisms.
1.2.4 The Role of Neuropeptides in the Brain
Over 70 genes in the mammalian genome encode for neuropeptides (16).
Neuropeptides are peptide molecules synthesized by neurons, are released in a regulated
manner and act on receptors present on other neurons. Compared to some classical
neurotransmitters, such as epinephrine, neuropeptides are large with nanomolar affinities for
their receptors. Neuropeptides can also diffuse over larger distances within the CNS than
some classical neurotransmitters. Indeed, neurotransmitters like glutamate have been shown
to have extrasynaptic effects. However, they are more likely to travel only as far as their
nearest neighbouring synapse. Glutamate, in particular, has been found to travel distances of
less than half a micrometer. In contrast, oxytocin released from neurons in the supraoptic
nucleus of the hypothalamus results in biologically relevant concentrations throughout the
anterior hypothalamus. In having high receptor binding affinity and the ability to affect
distant populations of neurons, neuropeptide release can mediate changes in neuronal activity
across multiple brain regions (17).
A growing number of neuropeptides and neurotransmitters have been implicated in
the regulation of feeding behavior in vivo. These neuropeptides are expressed in distinct
neuronal populations located in specific regions of the hypothalamus, including the arcuate,
paraventricular, and ventromedial nuclei (18).
7
1.2.5 The Melanocortin System
The melanocortin system is central to the neuronal control of energy homeostasis.
Here, the arcuate nucleus (ARC) of the hypothalamus is particularly important (19). Neurons
within the ARC are strategically located close to fenestrated capillaries at the base of the
hypothalamus such that they have access to circulating humoral signals (20). These neurons
are controlled by neurotransmitters that are released from neighbouring axons, express
receptors for metabolic hormones (20) and respond rapidly to nutritional cues (21).
At present, the mammalian central melanocortin system is defined as a collection of
CNS circuits that include: ARC neurons expressing hypothalamic neuropeptide Y (NPY) and
agouti-related peptide (AgRP) or proopiomelanocortin (POMC), brainstem POMC neurons
within the nucleus of the solitary tract (NTS) and downstream targets of these POMC and
AgRP neurons which express melanocortin 3 (MC3R) and melanocortin 4 (MC4R) receptors
(22).
1.2.6 Neuropeptide Y and Agouti-Related Peptide
Neuropeptides involved in food intake can be grouped into one of two categories:
orexigenic (appetite-stimulating) or anorexigenic (appetite-suppressing). The main
orexigenic neuron in the ARC is the NPY/AgRP neuron. Neuropeptide Y is a 36 amino acid
peptide that is expressed throughout the central nervous system (23), and has notably high
expression in the ARC (24). Agouti-related peptide is a 132 amino acid peptide that, unlike
NPY, is only found in the ARC. ARC NPY/AgRP neurons project to nearby hypothalamic
areas such as the paraventricular nucleus (PVN), dorsomedial nucleus (DMN), perifornical
8
area (PFA), lateral hypothalamic area (LHA) and the medial preoptic area (MPO), which are
integrative centers for the regulation of both feeding and energy expenditure (25).
NPY acts at multiple sites to increase food intake. Locally, NPY released from the
ARC acts to inhibit neighbouring POMC neurons by activation of Y1 and Y2 receptors (26).
NPY also acts on neurons in the PVN to stimulate food intake, and this effect appears to be
mediated by both Y1 and Y5 receptors (27, 28). However, unlike NPY, AgRP acts to increase
food intake by acting as an endogenous antagonist to the melanocortin 3 and 4 receptors (31).
This prevents the constitutive activity of these receptors (32), resulting in an inhibition of the
anorexigenic melanocortin pathway and an increase in food intake (Figure 1.2).
Insulin, among other hormones, regulates feeding and energy balance by modulating
the expression of these hypothalamic neuropeptides. Insulin may have anorexigenic effects
by increasing Pomc and decreasing Agrp gene expression (33), and this effect is mediated by
the phosphorylation of forkhead transcription factor 1 (FOXO1). FOXO1 is a transcription
factor that represses POMC gene expression and stimulates Agrp gene expression. Thus,
insulin-mediated phosphorylation of FOXO1 leads to its export from the nucleus which
relieves the repression on the POMC promoter (34). Concomitantly, FOXO1-induced
expression of Agrp in NPY/AgRP neurons is inhibited (35).
The importance of NPY and AgRP in the regulation of food intake and energy
homeostasis has been well documented. Central administration of either NPY (36) or AgRP
(29) increases food intake and body weight, and chronic administration results in obesity. A
single dose of AgRP results in an increase in food intake that is sustained for 7 days,
indicating its potency as an orexigenic neuropeptide (37). Inhibiting AgRP with arcuate-
9
Figure 1.2 NPY/AgRP and POMC neurons are directly regulated by insulin.
A representative diagram of the NPY/AgRP and POMC neurons, and how insulin regulates these neurons.
Insulin exerts its anorexigenic effects by increasing Pomc and decreasing AgRP gene expression. The
overall effect is a reduction in food intake and an increase in energy expenditure.
AgRP = agouti-related peptide
MC3R = melanocortin 3 receptor
MC4R = melanocortin 4 receptor
α-MSH = alpha-melanocyte stimulating
hormone
NPY = neuropeptide Y
Y1 Receptor = neuropeptide Y Y1 receptor
10
specific siRNA leads to decreases in both food intake and body weight (38). Furthermore,
ablation of these neurons in adult mice leads to extreme starvation (39, 40).
1.3 Glucose Sensing in Hypothalamic Neurons
1.3.1 Glucose Entry into the Brain and the Discovery of Glucose Sensing Neurons
Glucose is the primary energy substrate of the brain, and glucose metabolism accounts for
the majority of brain oxygen consumption. Stereospecific, but insulin-independent, GLUT-1
glucose transporters are highly expressed in brain capillary endothelial cells of the blood
brain barrier. GLUT-1 mediates the facilitated diffusion of glucose through the blood-brain
barrier, and can transport two to three times more glucose than is actually metabolized in the
brain (139). The stereospecificity of the GLUT-1 transporter allows D-glucose, but not L-
glucose, to pass into the brain.
Brain glucose varies depending on blood glucose, and declines to approximately 0.7
mM after an overnight fast. During peripheral hypoglycemia, hypothalamic glucose
concentrations have been shown to fall to as low as 0.3 mM (140). These and other studies
have indicated that hypothalamic glucose levels may range anywhere from 0.2 to 4.5 mM as
blood glucose levels vary from pathological hypoglycemia to hyperglycemia.
In 1964, two independent groups suggested the existence of glucose sensing neurons
(141, 142). In these studies, reciprocal changes in activity were measured in the ventromedial
hypothalamus (VMH) and lateral hypothalamus (regions referred to as the “satiety” and
“feeding” centers of the brain, respectively) following intravenous glucose or insulin
injections. In the VMH, glucose increased neuronal activity. In the lateral hypothalamus,
however, the opposite occurred. Later, Oomura et al. demonstrated that hypothalamic
11
neurons were directly regulated by glucose in vitro. This finding led to the terms “glucose
responsive” for neurons that increased their activity in response to increased glucose, and
“glucose sensitive” for neurons that decreased their activity in response to increased glucose.
Today, glucose sensing neurons are more commonly referred to as either glucose-exicted
(GE) or glucose-inhibited (GI) based on their physiological response to changes in
extracellular glucose (143).
1.3.2 Glucose-Excited Neurons
The expression of glucokinase (GK) (144) and ATP-sensitive potassium (KATP)
channels composed of Kir6.2 and SUR1 subunits (145) has led to the idea that GE neurons
sense changes in extracellular glucose concentrations via a mechanism that is similar to that
which operates in pancreatic β-cells. In this proposed model, increased glucose
concentrations are detected primarily through increased oxidation of glucose and generation
of ATP. The subsequent changes in electrical activity are mediated by closure of KATP
channels. Studies using transgenic POMC-green fluorescent protein (GFP) mice have shown
that ARC POMC neurons exhibit typical GE responses and express the KATP channel (146).
However, recent studies have suggested an additional population of GE neurons that
sense glucose independently of changes in KATP channel activity. A KATP channel-
independent glucose-sensing mechanism has been identified in a population of ARC GE
neurons, which is believed to involve cellular depolarization from the opening of a non-
specific cation channel in response to elevated glucose concentrations (147).
12
1.3.3 Glucose-Inhibited Neurons
In contrast to GE neurons, changes in AMP-activated protein kinase (AMPK) activity
are likely to mediate the inhibitory effects of glucose on ARC hypothalamic GI neurons
(148). AMPK is an evolutionarily conserved enzyme that acts as an intracellular energy
sensor to regulate fuel availability within a cell. AMPK is a heterotrimeric protein that
becomes activated allosterically by an increase in the intracellular AMP/ATP ratio. It has
been proposed that at low glucose concentrations, the rate of glucose uptake through GLUT3
and metabolism through GK and the glycolytic pathway are low. The resulting increase in
the AMP:ATP ratio would lead to activation of AMPK, which may then act to directly
phosphorylate and inactivate different ion channels leading to cellular depolarization (143).
While the mechanism of glucose-induced inhibition remains unclear, much more is
known about the physiological identities of these GI neurons. In the ARC, GI neurons were
found to co-express NPY and AgRP. Similarly, 94% of rat ARC neurons that were
stimulated by lowering extracellular glucose concentrations contained NPY
immunoreactivity (149). By switching extracellular glucose between 0.5 and 5 mM, 40% of
ARC NPY neurons were reversibly hyperpolarized and inhibited. Since NPY and AgRP are
orexigenic in nature, their co-localization in GI neurons implicates these neurons in the
mechanisms which lead to a stimulation of feeding.
1.4 Fatty Acid Sensing in Hypothalamic Neurons
1.4.1 Fatty Acids and Metabolic Physiology
The function of non-esterified fatty acids (NEFAs) was elucidated in the 1950’s
through the work of Vincent Dole (41) and Robert Gordon (42). Gordon demonstrated that
13
plasma NEFAs originate from adipose tissues, and elucidated their use by tissues such as the
liver and myocardium. We now understand that NEFAs are the primary fuel for most tissues
under fasting conditions (43). The release of NEFAs into the circulation results partly from
the hydrolysis of triacylglycerol-rich lipids via the action of lipoprotein lipase (LPL) (43). In
addition to being an important source of energy, NEFAs are also necessary for membrane
lipid synthesis and lipid signaling (44). Although mostly bound to albumin, NEFA turnover
is fast. The circulating half-life of NEFAs is only 3-4 minutes (43). In the fasting state,
plasma NEFAs arise almost entirely from the hydrolysis of trigylcerides (TG) in adipocytes
(45). However, after a meal, there is an additional source of plasma NEFA. LPL in the
capillaries of adipose tissue hydrolyzes circulating TG, which constitutes much of the dietary
fat carried in chylomicrons. Though fatty acids thereafter become taken up by adipocytes for
storage, there is always a proportion that escapes and joins the plasma NEFA pool (46).
Therefore, the plasma NEFA pool composition changes in accordance with the composition
of meal fat (47).
Fat mobilization is rapidly suppressed by insulin. Therefore, plasma NEFA
concentrations fall after any meal containing carbohydrates. Typical plasma NEFA
concentrations range from 300-600 µmol/L in an overnight fasting state to approximately
1,300 µmol/L after a 72 hour fast (48).
1.4.2 Blood-Brain Barrier Permeability, Fatty Acid Uptake and Metabolic Fates
Once in the plasma, free fatty acids are bound by the carrier protein albumin.
Albumin increases the solubility of these FFAs and facilitates their transport across
membranes (44). To cross the blood-brain barrier, FFAs readily desorb from albumin and are
14
rapidly taken up by a “flip-flop” diffusion process (49) and/or transport proteins. Transport
proteins include CD36, fatty acid transport protein (FATP) and plasma membrane fatty acid
binding protein (FABP) (50). Upon entry into the cell, FFAs become coupled to FABPs,
which carry FFAs from the plasma membrane to their target organelles.
After cellular uptake, fatty acids become rapidly esterified to a fatty acyl-coenzyme A
(fatty acyl-CoA). This reaction is catalyzed by the enzyme acyl-CoA synthetase (51). In this
activated aycl-CoA form, fatty acids can be (i) degraded by mitochondrial β-oxidation to
provide cellular energy, (ii) esterified to membrane lipids or (iii) enter the sphingolipid
pathway and contribute to the generation of ceramide metabolites (Figure 1.3).
1.4.3 Fatty Acids as Signaling Molecules in the Hypothalamus
Fatty acyl-CoA’s and the pathways regulating fatty acyl-CoA metabolism have been
implicated in the hypothalamic control of feeding behavior and energy homeostasis. One
hypothesis is that circulating lipids regulate feeding behavior by generating an increase in the
hypothalamic fatty acyl-CoA pool. In turn, these fatty acyl-CoA’s signal an energy surplus
within the hypothalamus, which activates neuronal circuits to decrease both food intake and
liver glucose production (52). Indeed, intracerebroventricular (icv) administration of the
monounsaturated fatty acid oleic acid is sufficient to inhibit food intake and liver glucose
production. Furthermore, icv oleic acid inhibits the expression of orexigenic NPY and AgRP
in the hypothalamus (53).
Given these findings, it was hypothesized that similar metabolic and behavioral
effects would be seen by increasing fatty acyl-CoA availability. Under genetic or
pharmacological inhibition of hypothalamic CPT1 (an enzyme which facilitates the transport
15
Fig. 1.3 Metabolic fates of palmitate upon entering the cell.
Once palmitate enters the cell, it becomes “primed” in order to cross the mitochondrial membrane.
This occurs in the peroxisome, where peroxisomal acyl-CoA synthetase catalyzes the reaction
between the fatty acid and CoA. The resultant palmitoyl-CoA can then: 1) enter the mitochondria
where it undergoes β-oxidation, 2) participate in protein palmitoylation via the actions of protein
acyltransferase or 3) contribute towards de novo ceramide synthesis.
16
of fatty acyl-CoA molecules into the mitochondria for β-oxidation), the concentration of
hypothalamic fatty acyl-CoA’s increased, whereas the expression of orexigenic NPY and
AgRP decreased (54). Therefore, these data lend support to the idea that fatty acids and the
availability of fatty acyl-CoAs are important components of hypothalamic lipid sensing.
1.5 Obesity and Hypothalamic Inflammation
1.5.1 High-Fat Feeding is Associated with Inflammation
Excessive caloric intake is a primary risk factor for the development of obesity.
Epidemiological studies have shown that individuals consuming high fat diets are
particularly prone to gaining body mass (55). In peripheral tissues, the deleterious metabolic
consequences of obesity arise, in part, from cellular inflammation triggered by this nutrient
excess. Excess visceral adiposity is accompanied by chronic low grade inflammation in the
liver, adipose tissue, skeletal muscle and vasculature. This inflammation is associated with
increased circulating levels of pro-inflammatory cytokines (56). Circulating saturated fatty
acids are also capable of triggering Toll-like receptor (TLR) signaling, which results in
subsequent activation of intracellular inflammatory signals such as inhibitor of kB-kinase-β
(IKKβ)/nuclear factor-kB (NF-κB) and c-Jun N-terminal kinase (JNK) (57). The end result is
a vicious cycle of inflammation that produces progressive, systemic metabolic impairment.
In 2005, evidence emerged that inflammatory changes could be detected in the brains
of high fat diet-fed animals. A 20-week HFD-feeding study found increased NF-κB signaling
in the rat cerebral cortex (58). Honing in on the hypothalamus, De Souza et al. (59) tested the
hypothesis that high fat consumption could modulate gene expression in the hypothalamus.
Using a macroarray, the expression of more than 1,000 hypothalamic genes was
17
simultaneously measured. Of the 1,000 genes examined, more than 15% were modulated by
diet. Grouping the genes based on function revealed that inflammatory genes were most
affected after 16 weeks of HFD feeding (59).
1.5.2 Evidence that Hypothalamic Inflammation Contributes to HFD-Induced Obesity
Consistent with a role for hypothalamic inflammation in diet-induced obesity,
neuron-specific disruption of TLR4 or IKKβ/NF-κB pathways protected against diet-induced
obesity and its associated metabolic consequences (60). Viral deletion of IKKβ or over-
expression of a dominant-negative IKKβ isoform in the mediobasal hypothalamus also
reduced food intake and weight gain during HFD feeding (60). Moreover, in genetically
normal animals, central infusion of an IKKβ inhibitor or antibodies to TLR4 can reduce food
intake in diet-induced obese (DIO) mice (61). Taken together, these studies demonstrate the
causal role of hypothalamic inflammation in HFD-induced weight gain.
Complementing these findings is the fact that augmented hypothalamic inflammation
is associated with HFD-induced obesity. For example, neuronal expression of a constitutively
active IKKβ isoform increases food intake (60). Furthermore, infusion of the cytokine IL-4
directly into the brain of HFD-fed rats exerts a pro-inflammatory effect on the hypothalamus
that exacerbates weight gain in an IKKβ-dependent manner (62). These data suggest that
hypothalamic inflammation is both necessary and sufficient for initial and sustained weight
gain during HFD feeding.
Yet, if hypothalamic inflammation is to be implicated in obesity pathogenesis, it must
occur prior to obesity onset. Indeed, hypothalamic inflammation is observed weeks before
18
peripheral cytokines are produced in the liver and adipose tissue, and before alterations in
body weight occur (63).
1.5.3 Palmitate as a Mediator of Hypothalamic Inflammation
Whether hypothalamic inflammation is a consequence of excess caloric intake
irrespective of diet composition has been the subject of considerable debate. One mechanism
that has received attention is the ability of saturated versus unsaturated fatty acids to activate
TLR4/NF-κB signaling. A predominant saturated fatty acid in our diet, palmitic acid (16:0),
is found in high concentrations in all animal products and accounts for approximately 20-
30% of the total FFAs in humans. Palmitic acid enters the brain linearly with time and is
rapidly incorporated into brain lipids (64). Many studies that have investigated the role of
FFAs in HFD-induced hypothalamic inflammation have utilized palmitic acid. Recent work
demonstrates that saturated fatty acid palmitate (16:0) induces NF-κB signaling through a
TLR4-dependent mechanism when administered in neuronal cell culture and after infusion
into the brain (65). Signaling through NF-κB leads to the induction of cytokine gene
expression, which causes local levels of TNF-α, IL-1β and IL-6 to rise and exacerbate the
inflammatory state (Figure 1.4) (66).
1.6 Obesity and Hypothalamic Insulin Resistance
1.6.1 Obesity, Diabetes and Insulin Resistance
Currently, more than one third of U.S. adults are obese (which is defined as having a
BMI >30 kg/m2) and over 11% of individuals aged 20 or over have diabetes (67). Due to the
strong association between T2DM and obesity, Zimmet et al. coined the term “diabesity”
(68). However, only 20% of obese individuals develop T2DM, and this is thought to be due
19
Fig. 1.4 Activation of the IKKβ/NF-κB pathway leads to inflammation and impaired insulin signaling.
Activation of TLR4 or TNF-α receptor by palmitate and TNF-α, respectively, stimulates downstream NF-
κB and AP-1 transcription factors to upregulate gene expression of pro-inflammatory cytokines. Insulin
signaling is inhibited by chronic receptor stimulation by insulin itself or by stimulation of the TLR4 and/or
TNF-α receptors. IKKβ and JNK, in particular, can inhibit insulin signaling by phosphorylating serine
residues on IRS proteins.
AP-1 = activator protein 1
IKKβ = inhibitor of IkappaB kinase beta
Ins = insulin
IR = insulin receptor
IRS = insulin receptor substrate protein
JNK = c-Jun N-terminal kinase
Pal = palmitate
TNF = tumor necrosis factor
TLR4 = toll-like receptor 4
TNFR = tumor necrosis factor receptor
TAK1 = transforming growth factor-β-activated kinase 1
NF-κB = nuclear factor kappa B
20
to a compensatory response by pancreatic β-cells to increase insulin secretion. Therefore,
T2DM involves both decreased insulin sensitivity and a loss of compensatory insulin
secretion. Insulin resistance, then, is defined as a diminished cellular response to insulin,
resulting in the inability to increase glucose uptake leading to increased blood glucose (69).
Obesity results from an imbalance between energy intake and energy expenditure.
The result is adipocyte hypertrophy, along with increased lipolysis (70). Increased adiposity
can lead to insulin resistance through increased adipocyte-derived FFAs and increased
adipokines (71). Excess FFAs become stored in non-adipose cells such as muscle, where they
may be catabolized into lipid metabolites such as fatty acyl-CoAs, diacylglycerol (DAG),
triacylglycerol (TAG) and ceramide. These lipid metabolites are capable of inhibiting insulin
signal transduction leading to insulin resistance (71). Moreover, in an obese state, there is an
increase in peripherally-derived pro-inflammatory cytokines such as tumor necrosis factor-
alpha (TNF-α), which have also been shown to induce insulin resistance in mice and humans
(72). Another contributing factor to the development of insulin resistance is
hyperinsulinemia, which will be discussed in the following section. Finally, impaired insulin
signaling has the ability to potentiate obesity pathogenesis due to the importance of central
insulin action in regulating energy balance. Therefore, the maintenance of proper insulin
action is critical for maintaining energy homeostasis.
1.6.2 Hyperinsulinemia and the Development of Insulin Resistance
Insulin resistance is a hallmark feature of obesity. There are numerous etiologies for
insulin resistance, including lipotoxicity, inflammation and hyperinsulinemia (73).
Hyperinsulinemia reflects the compensation by insulin-secreting β-cells to systemic insulin
21
resistance. In vivo studies indicate that prolonged exposure to high levels of insulin can lead
to insulin resistance (74). Indeed, plasma insulin levels are increased in obese states and this
increase occurs prior to a reduction in insulin sensitivity (75). At the cellular level,
hyperinsulinemia impairs insulin signal transduction through homologous desensitization.
The insulin receptor itself is involved in negative feedback involving a reduction in
(i) receptor affinity, (ii) the number of receptors expressed on the cell surface and (iii) the
effectiveness of the receptor as a transmitter of stimulatory signals (76). Continual exposure
to insulin can also lead to serine phosphorylation of downstream IRS proteins, which reduces
its ability to activate downstream elements in the insulin signaling pathway (76).
1.6.3 High Fat Feeding and Neuronal Insulin Resistance
The growing trend towards a more sedentary lifestyle combined with the
consumption of fat-rich foods play an important role in the current obesity epidemic (34).
Consumption of HFD for as little as 72 hours is sufficient to reduce hypothalamic insulin
sensitivity in rats (77). Elevated saturated fatty acids not only increase body weight, but also
chronically reduce hypothalamic insulin sensitivity (77). At a molecular level, saturated fatty
acids such as palmitate cross the blood-brain barrier and accumulate in the hypothalamus.
Here, they activate pro-inflammatory signaling pathways including toll-like receptor 4
(TLR4) signaling, resulting in central insulin resistance (65).
Palmitate-mediated central insulin resistance is due, at least in part, to the activation
of protein kinase Cθ (PKCθ). Subsequent translocation of PKCθ to the cell membrane
prevents insulin-mediated activation of PI3K via direct interaction with insulin receptor and
insulin receptor substrate proteins (78). On the other hand, palmitate can also activate NF-κB,
22
which subsequently induces suppressor of cytokine signaling (SOCS) 3 expression (60).
SOCS3 is one of the principal negative regulators of insulin signaling, interfering with
insulin-mediated phosphorylation of IR and its downstream molecules. It also targets IRS
proteins for proteasomal degradation (79). Elevated NF-κB signaling also triggers
endoplasmic reticulum stress leading to increased activity of c-Jun N-terminal kinase (JNK).
In turn, JNK mediates inhibitory phosphorylation events on IRS serine residues which
contribute to the development of insulin resistance (80,81).
During obesity progression, elevated levels of pro-inflammatory cytokines exacerbate
central insulin resistance by further activating NF-κB and JNK signaling. TNF-α, like
palmitate, can induce expression of protein tyrosine phosphatase 1B (PTP1B), potentially
through transactivation of NF-κB (82). Elevated levels of PTP1B in the ARC, as seen after
20 weeks of HFD feeding, inhibit insulin signaling by direct dephosphorylation of the IR
(Fig. 1.5) (83).
1.6.4 FFA Metabolism and Insulin Resistance
Dysregulated FFA metabolism is thought to play a causal role in the development of insulin
resistance (84). The large majority of FFAs enter the glycerolipid pathway, where they
become substrates for membrane glycerophospholipids and TAG, the primary form of stored
fat (86). Glycerol-3-phosphate acyltransferase (GPAT) is the enzyme that regulates entrance
of FFAs into this pathway, and it transfers the acyl group from fatty acyl-CoA to glycerol-3-
phosphate (86). The saturated fatty acid palmitate increases intracellular levels of DAG, the
precursor to TAG, and TAG itself in rat islets (87). Interestingly, the GPAT knockout mouse
is protected from insulin resistance on a HF diet (88).
23
Fig. 1.5 Mechanisms of palmitate-mediated inhibition of insulin signaling.
Palmitate has been shown to inhibit insulin signaling through a variety of mechanisms. 1) Palmitate may
enter the cell and contribute to an increase in de novo ceramide synthesis. Ceramides are known to interact
with and activate the phosphatase enzyme PP2A. PP2A dephosphorylates Akt, leading to impaired insulin
signaling. 2) Palmitate can increase intracellular levels of diacylglycerol, leading to activation of PKCθ and
subsequent serine phosphorylation of IRS-1. 3) Palmitate (and inflammation) induces the expression of
PTP1B, which is a protein tyrosine phosphatase enzyme. PTP1B inhibits insulin signaling by removing
phosphate groups from the tyrosine residues of the activated insulin receptor.
AKT = protein kinase B
DAG = diacylglycerol
ER = endoplasmic reticulum
Pal = palmitate
Pal-CoA = palmitoyl-CoA
PKCθ = protein kinase C θ
PP2A = protein phosphatase 2A
PTP1B = protein tyrosine
phosphatase 1B
24
Rates of ceramide synthesis depend on the availability of long-chain saturated fatty
acids, which participate in the initial rate-limiting step of de novo ceramide synthesis (86). In
this reaction, serine palmitoyltransferase (SPT) catalyzes the condensation of palmitoyl-CoA
and serine to produce 3-ketosphinganine. Subsequent reactions lead to the eventual synthesis
of ceramide, which serves as a precursor for more complex sphingolipids (86). Ceramides
with different fatty acid and long-chain base compositions can be formed in different
compartments or membranes of the cell, each with potentially distinct functions.
Interestingly, ceramide levels are elevated in rodent and human insulin-resistant tissues (89).
As demonstrated in numerous reports, elevated ceramide levels may inhibit insulin-
stimulated glucose uptake, GLUT4 translocation and/or glycogen synthesis (90). This
dysregulation of insulin signaling has been linked to ceramide-mediated regulation of IRS-1
and Akt/PKB.
Three independent groups found that treating cultured cells with short-chain ceramide
analogs blocked insulin-stimulated tyrosine phosphorylation of IRS-1 and its subsequent
activation of PI3K (91). These groups proposed that ceramide may promote the
phosphorylation of IRS-1 on inhibitory serine/threonine residues. In various cell types,
ceramides have been shown to activate extracellular signal-regulated kinase 2 (ERK2), p38,
JNK and IkB kinases (IKKs) (92), which have been implicated in serine/threonine
phosphorylation of IRS-1 (93).
Many groups have demonstrated that ceramide also inhibits phosphorylation and
activation of Akt/PKB. It is now understood that ceramide inhibits activation of Akt/PKB
through two distinct mechanisms. First, ceramide promotes the dephosphorylation of
Akt/PKB through direct activation of protein phosphatase 2A (PP2A) (94). Indeed, the PP2A
25
inhibitor okadaic acid was sufficient to prevent the inhibitory ceramide effects on Akt/PKB
in C2C12 myotubes and brown adipocytes (95, 96). Second, ceramide may activate PKCζ,
which inhibits Akt/PKB translocation to the membrane by phosphorylating threonine-34
(97).
1.7 Cell Model
1.7.1 The Need for Cell Lines
In vivo experimentation is important for determining the overall function of a
molecule (i.e. hormone, receptor or structural protein). Indeed, the use of animal models has
enhanced our basic understanding of physiological processes, such as energy homeostasis.
These studies have elucidated the role of the brain in overall metabolism, and have triggered
the development of brain-specific and neuron-specific mouse models. Yet, despite new
technologies allowing for closer examination of intracellular workings, animal
experimentation has its limits. This is especially true in the context of the hypothalamus,
where a collection of cell phenotypes exist. Therefore, classical in vivo approaches cannot
establish the molecular mechanisms involved in gene regulation and cellular signaling.
Moreover, it is difficult to determine the direct actions of agents, such as nutrients or
hormones, on specific cell types. Given these limitations, researchers have turned to the use
of cell lines (98).
Cell lines allow for experimentation with homogeneous populations of cells in a
controlled environment. However, one cannot state for certain that cell lines function exactly
as the native cells would. For this reason, caution must be taken before extrapolating findings
from cell lines to an in vivo model. When working with neuronal cell lines, it is particularly
26
important to remember that these models lack the complexity and integrated network of
neurons found in vivo. Regardless, recent studies looking at molecular events in vitro have
found that the results from cell lines mirror that of in vivo studies (98).
Basic tissue culture techniques were established in 1885 by Wilhelm Roux, and it was
not until 1940 that the first immortal cell line was developed (99). Since that time, cell lines
have been produced from many different tissues although the first attempt at immortalizing
neurons was not performed until 1974 by Shaw et al. (100). Shaw et al. transfected primary
hypothalamic cells from embryonic mice with simian virus 40 T-Ag to create an
immortalized cell population labeled HT9. Years later, Cepko et al. developed retroviral
shuttle vectors which would allow researchers to retrovirally infect primary cells with an
immortalizing oncogene and selectively propagate them (101).
1.7.2 Adult Hypothalamic Cell Lines (mHypoA-xx)
Non-transformed primary hypothalamic cultures are difficult to maintain, have a short
lifespan and represent a heterogeneous population of neurons. Contrarily, immortalized,
clonal cell lines represent an unlimited and homogeneous population of specific neuronal cell
types. Since the hypothalamus contains a wide range of cell types, Belsham et al. recognized
the need for mouse cell lines representative of many unique hypothalamic neurons. Belsham
et al. initially developed 38 embryonic, clonal hypothalamic mouse cell lines (98). However,
to understand key molecular mechanisms involved in adult neuroendocrine cells, the
Belsham group also immortalized adult hypothalamic cell models.
In order to immortalize the adult neurons, cells were treated with ciliary neurotrophic
factor (CNTF) to induce cell proliferation. This would render the cell cultures amenable to
27
retroviral transfer of the SV40 T-antigen oncogene (Figure 1.6) (102). Over 50 adult mouse
cell lines were eventually established, and labeled in the form mHypoA-‘clone number’. Like
the embryonic cell lines before them, the adult cell lines also express mature neuronal
markers, display typical neuronal morphology and have been characterized for the expression
of necessary neuropeptides and receptors. The hypothalamic cell lines made available from
our lab and others allow for the study of molecular events involved in nutrient sensing in
distinct neuronal populations. These novel, representative cell models put us in an
advantageous position to determine the direct effects of nutrients (i.e. palmitate) on
neuropeptide gene expression and signaling events in neuropeptide-expressing neurons.
1.7.3 mHypoA-NPY/GFP Cell Line
To immortalize NPY/AgRP-expressing neurons from the adult hypothalamus, the
Belsham group dissected hypothalamii from NPY-GFP transgenic mice and immortalized as
described above. NPY/AgRP neurons were then selected using flow cytometry. The NPY-
GFP cell line has been thoroughly characterized using RT-PCR, ICC and NPY secretion
assays, and the phenotypic characterization is described in Table 1.1. In this thesis, the
mHypoA-NPY/GFP cell model is used to describe palmitate-mediated regulation of AgRP
gene expression and to elucidate the effects of palmitate on signaling events in representative
NPY/AgRP-expressing neurons.
1.8 Hypothesis and Aims
Neuronal circuits within the hypothalamus form the homeostatic control mechanism
that controls food intake and energy balance. It has been well established that coordinated
regulation
28
1) 2)
3) 4) 5)
Fig. 1.6 Generation of the mHypoA-NPY/GFP cell line.
Adult NPY/GFP-expressing transgenic mice were generated (1). Cells were harvested from the
GFP-expressing mouse hypothalamus (2). These cells were then treated with CNTF to induce
neurogenesis, and transfected with SV-40 T antigen for immortalization (3). Cells were FAC sorted
for GFP fluorescence with greater than 95% purity (4). The fluorescent cells represent the cells of
interest, mHypoA-NPY/GFP (5).
29
of neuropeptide gene expression from the hypothalamus is critical to maintain normal energy
homeostasis. Disturbances at the hypothalamic level may lead to metabolic disease. A
leading contributor to diet-induced obesity and T2DM is hypothalamic inflammation and
insulin resistance in response to saturated fatty acids consumed in the diet. In fact,
hypothalamic inflammation occurs weeks before peripheral cytokines are produced and
before alterations in body weight occur. Several molecules and pathways have been
identified as mediators of hypothalamic inflammation during HFD feeding, including JNK,
IKKβ and TLR4. JNK and IKKβ, in particular, can inhibit insulin signaling through
induction of SOCS3 signaling or serine phosphorylation of insulin receptor substrate. For
these reasons, hypothalamic inflammation is an important new target for obesity therapeutics.
Research has since focused on understanding how HFD induces hypothalamic
inflammation and insulin resistance. Palmitate (16:0), a non-esterified saturated fatty acid
which exists at high levels in the plasma of obese individuals, has received considerable
attention due to its ability to activate TLR4/NF-κB signaling. Indeed, icv injection of
palmitate has been shown to attenuate hypothalamic insulin signaling and increase IKKβ
activity.
Though animal models have proven invaluable in establishing our basic understanding
of energy homeostasis, the specific molecular events involved in nutrient (particularly fat)
sensing in distinct neuronal populations remain largely unknown. However, the Belsham
laboratory has generated several immortalized, hypothalamic neuronal cell lines from
primary fetal and adult hypothalamic neuronal cell culture. These representative cell models
enable us to determine the direct effects of nutrients (i.e. palmitate) on neuropeptide gene
expression and signaling events in neuropeptide-expressing neurons. This thesis involved the
30
use of the mHypoA-NPY/GFP cell line, which is a pure population of representative NPY
neurons from the entire hypothalamus. We used this cell line to study the effects of palmitate
on inflammatory status, AgRP gene expression and insulin signaling in representative
NPY/AgRP neurons.
It was therefore hypothesized that palmitate would alter cellular function in
NPY/AgRP neurons. Treatment of mHypoA-NPY/GFP cells with palmitate would: (i)
induce a state of inflammation, (ii) alter AgRP gene expression and (iii) alter insulin
signal transduction.
Aim #1: Determine whether palmitate can alter inflammatory status and AgRP gene
expression in mHypoA-NPY/GFP neurons. Whether changes (if any) in AgRP gene
expression are palmitate metabolism-dependent will also be determined. These results are
presented in section 3.1 and 3.3.
Aim #2: Determine whether TNF-α, a pro-inflammatory surrogate of palmitate, can
alter inflammatory status and AgRP gene expression in mHypoA-NPY/GFP neurons. These
results are presented in section 3.2.
Aim #3: Determine whether palmitate induces the phosphorylation/activation of
ERK1/2, JNK and/or p38 MAPK in mHypoA-NPY/GFP neurons. These results are presented
in section 3.4.
Aim #4: Determine whether prolonged exposure to insulin or palmitate will have an
effect on insulin signaling in mHypoA-NPY/GFP neurons. These results are presented in
section 3.5.
31
Table 1.1 Characterization of the mHypoA-NPY/GFP cell line via immunofluorescence
and semi-quantitative PCR.
(Screening data courtesy of Jennifer Chalmers and Prasad Dalvi)
mRNA mHypoA-NPY/GFP
Enzyme mRNA
CPT1a and c +
Na+/K
+ ATPase +
Transporter, Channel and Receptor mRNA
GLUT 1,3,4,5,8 +
GLUT 2 -
IR +
GPR120 +
ObRb +
TLR4 +
Neuropeptide and Cytokine mRNA
AgRP +
NPY +
POMC -
TNF-α +
IL-6 +
32
Chapter 2
Materials and Methods
33
2. Materials and Methods
2.1 Cell Culture and Reagents
The hypothalamic cell line from the adult mouse, mHypoA-NPY/GFP, was isolated
and immortalized as previously described (103). mHypoA-NPY/GFP cells were grown to
confluency and maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Sigma, St.
Louis MO, USA) supplemented with 5% fetal bovine serum (HyClone Laboratories, Logan,
UT) and 1% penicillin/streptomycin (Life Technologies Inc., Burlington, Canada) at 37°C in
an atmosphere of 5% CO2. Cells were then seeded onto 60 mm culture plates 24 hours prior
to treatments. Sodium palmitate, TNF-α and methyl palmitate were obtained from Sigma-
Aldrich (Oakville, Canada). Insulin was purchased from Novo Nordisk Canada Inc.
(Mississauga, Canada).
2.2 Palmitate Preparation
A 25 mM stock of palmitate was first prepared by dissolving approximately 6.9 mg of
sodium palmitate (Sigma) in 1 mL of Hypure water (Thermo Scientific, Rockford, IL, USA)
and heated to 60°C. Upon treatment, the media in each 60 mm culture plate was replaced
entirely with 3 mL of fresh treatment media. To achieve a working concentration of 25 μM
palmitate in the treatment media (DMEM, 5% FBS, 1% penicillin/streptomycin), 3 μL of
stock palmitate was added per 3 mL treatment media.
2.3 TNF-α Preparation
Stock TNF-α powder (10 µg) was dissolved in 1 mL of Hypure H2O. 50 uL aliquots
of 10 µg/mL TNF-α were then stored in a -20°C freezer. The day before treating cells with
TNF-α, media was changed to 2.5 mL low glucose, 5% FBS, 1% penicillin/streptomycin
34
media. To achieve a working concentration of 50 ng/mL TNF-α in the culture plates, 15 μL
of stock TNF-α was added per 500 μL serum-free DMEM and added to each culture plate on
the day of treatment.
2.4 Insulin Preparation
Insulin (600 µM) was first diluted with 1x PBS to a stock concentration of either 100
µM or 10 µM in 0.6 mL eppendorf tubes. Working concentrations of 100 nM or 10 nM
insulin were achieved by adding 3 μL of stock insulin per 500 μL serum-free media and
added to cell culture dishes already containing 2.5 mL serum-free media.
2.5 Quantitative RT-PCR
Total RNA was isolated from mHypoA-NPY/GFP cells at the indicated time points
using the guanidinium thiocyanate phenol chloroform extraction method, as previously
described (104). RNA concentrations, and their accompanying purity ratios, were determined
using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Nepean, Ontario, Canada).
Contaminating DNA was removed from all RNA samples using Turbo DNAase (Ambion,
Austin, TX, USA) treatment (1 hr, 37°C). RNA was then reverse-transcribed using the High
Capacity cDNA Reverse Transcriptase kit (Applied Biosystems, Streetsville, Ontario,
Canada). 50 ng of cDNA was loaded per sample well and amplified using an Applied
Biosystems Prism 7000 Real-Time PCR machine together with gene-specific primers (Cell
Signaling Technology Inc., Danvers, MA, USA) and SYBR green master mix. Each sample
was run in triplicate according to the following cycle sequence: 2 minutes at 50°C, 10
minutes at 95°C; 40 cycles of 15 seconds at 95°C, 1 minute at 60°C; 15 seconds at 95°C, 15
35
seconds at 60°C, and 15 seconds at 95°C. Quantitative reverse transcriptase PCR data was
then quantified using the standard curve method and normalized to histone 3a.
2.6 Western Blot Analysis
Total protein was harvested using 1x cell lysis buffer (Cell Signaling) supplemented
with 1 mM PMSF, 1% phosphatase inhibitor cocktail 2 (Sigma) and 1% protease inhibitor.
The soluble fraction was isolated after centrifugation at 14,000 rpm for 10 minutes at 4°C.
Protein was then quantified using a BCA protein assay kit (Thermo Scientific) according to
the manufacturer’s instructions, and lysates were resolved on 8% polyacrylamide gels and
transferred onto Immobilon-P PVDF membrane (Bio-Rad, Hercules, CA, USA). Membranes
were blocked in 5% milk in Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 1 hour,
followed by primary antibody incubation overnight at 4°C. Membranes were washed with
TBS-T before and after incubation with goat anti-rabbit HRP secondary antibody (Cell
Signaling) for one hour. Protein was visualized using a Kodak Image Station 2000R
(Eastman Kodak Company, Rochester, NY, USA) and band intensity was quantified using
Kodak 1D Image Analysis Software 3.6. Primary antibodies used for western blotting include
Gβ, phospho-AKT, AKT, phospho-p44/42 MAPK (ERK1/2), phospho-JNK and phospho-
p38 MAPK. All primary antibodies were obtained from Cell Signaling.
In section 3.4, mHypoA-NPY/GFP neurons were treated with palmitate (25 µM) or
vehicle alone over a 60 minute time course and protein was harvested at 5, 15, 30 and 60
minute time points. For insulin resistance experiments, cells were pre-treated with either 100
nM insulin or 25 µM palmitate for 24 hours before insulin treatment media was changed to
serum-free DMEM for one hour. Cells were then re-challenged with 10 nM insulin and
36
protein was isolated after 15 minutes. In the insulin resistance studies, phospho-proteins were
normalized to total protein. However, for the signaling experiments, phospho-proteins were
normalized to the loading control Gβ. Although total protein comparisons are ideal, our
laboratory has found in previous studies that Gβ is a reliable indicator of loading status.
2.7 Statistical Analysis
Data are presented as the mean ± the standard error of the mean (SEM). All statistics
were calculated using Graphpad Prism 6.0 (San Diego, CA, USA). Groups were compared
using two-way analysis of variance (ANOVA) with Tukey’s test for post hoc comparisons. A
minimum of three experimental replicates were performed for each experiment. Significance
is denoted by *p <0.05, **p <0.01 and ***p <0.001.
37
Chapter 3
Results
38
3. Results
3.1 Palmitate elicits an inflammatory response and upregulates Agrp gene expression in
mHypoA-NPY/GFP neurons
Animal models of diet-induced obesity exhibit inflammation in hypothalamic areas
critical for energy homeostasis. This is generally defined as the activation of pro-
inflammatory signaling within neurons and glia (56). While systemic levels of pro-
inflammatory cytokines, such as IL-6 and TNF-α, are also elevated in the obese state (105),
hypothalamic inflammation has been shown to occur weeks before peripheral cytokines are
even produced (63). Long-chain saturated fatty acids, in particular, have been shown to
activate toll-like receptor 4 signaling to induce local cytokine expression in these neurons
(65). Indeed, mice with neuron-specific deletion of IKKβ are resistant to the effects of a HFD
to cause obesity and central insulin resistance (60). Such findings suggest a role for insulin
resistance induced by hypothalamic inflammation in the mechanism whereby HF feeding
leads to obesity. However, the specific hypothalamic neuronal cell types that trigger this
inflammation in response to HF feeding remain unknown.
To determine whether the saturated fatty acid palmitate can elicit an inflammatory
response in mHypoA-NPY/GFP neurons, qRT-PCR was used to assess relative changes in
mRNA levels of NF-κB (p105 subunit) and IkBα following treatment with 25 µM palmitate.
Treatment with 25 µM palmitate for 2 hours was sufficient to increase NF-κB mRNA levels
relative to control (n=3; P<0.05; Fig. 3.1A) in this cell line. Within 4 hours, this dose of
palmitate also significantly increased IkBα mRNA levels (n=3; P<0.01; Fig. 3.1B).
39
While changes in AgRP gene expression are likely following the establishment of
insulin resistance in NPY/AgRP neurons, the ability of fatty acids to directly modulate AgRP
gene expression is unknown. Therefore, to determine whether AgRP gene expression could
be modulated by the saturated fatty acid palmitate in mHypoA-NPY/GFP neurons, qRT-PCR
was used to assess the regulation of AgRP mRNA levels upon exposure to palmitate.
Treatment of mHypoA-NPY/GFP neurons with palmitate (25-50 µM) led to an increase in
AgRP mRNA levels at 24 hours (n=3-4; P<0.05; Fig. 3.1 E). However, high levels of
saturated fatty acids can be toxic to the neurons. To this end, we evaluated the toxicity of the
doses used. In particular, we examined the susceptibility of the mHypoA-NPY/GFP neurons
to apoptosis by measuring the ratio of Bax/Bcl2 mRNA. While 50 µM palmitate resulted in a
significant increase in Bax/Bcl2 mRNA at all time points examined (n=3; P<0.05; Fig.
3.1D), 25 µM palmitate did not alter this ratio at any time (n=4; Fig. 3.1C). Since the
susceptibility to apoptosis was minimized with 25 µM palmitate, it would be the
concentration used in all subsequent studies.
3.2 TNF-α, a pro-inflammatory surrogate of palmitate, also upregulates AgRP gene
expression in mHypoA-NPY/GFP neurons
Given palmitate’s ability to activate TLR4 and downstream IKKβ/NF-κB signaling, it is
likely that palmitate also induces expression of the pro-inflammatory cytokine TNF-α. TNF-
α is a pro-inflammatory cytokine that drives tissue inflammation, at least in part, through
activation of the classic IKKβ/NF-κB inflammatory signaling pathway. Activation of the
transcription factor NF-κB leads to further expression of pro-inflammatory cytokines, such as
TNF-α, IL-1β and IL-6. However, it is not known whether TNF-α can directly regulate AgRP
gene expression in NPY/AgRP neurons. Therefore, mHypoA-NPY/GFP neurons were
40
3.3 Palmitate-mediated regulation of AgRP gene expression is metabolism-dependent
Time (h)
Bax/B
cl2
/His
ton
e m
RN
A
4h 24h0.0
0.5
1.0
1.5
2.0
Time (h)
Bax/B
cl2
/His
ton
e m
RN
A
4h 24h0.0
0.5
1.0
1.5
2.0
*
*
Time (h)
NF
-kB
/His
ton
e m
RN
A
1 2 4 240.0
0.5
1.0
1.5 *
Time (h)
IkB
/His
ton
e m
RN
A
1 2 4 240.0
0.5
1.0
1.5
2.0
2.5**
4h 24h0.0
0.5
1.0
1.5
2.0
Time (h)
Ag
RP
/His
ton
e m
RN
A
H2O
25 uM Palmitate*50 uM Palmitate
*
Fig. 3.1 Saturated fatty acid palmitate upregulates pro-inflammatory and Agrp gene expression.
Top: mHypoA-NPY/GFP neurons were treated with 25 µM palmitate (grey) or vehicle (black), RNA was isolated from 1 to 24
hours and analyzed using quantitative real-time PCR with primers for NF-κB (A) or IκBα (B). Treatment with 25 µM palmitate
resulted in a significant increase in NF-κB transcript levels at 2 hours, and a significant increase in IκBα transcript levels at 4
hours. Data was normalized to histone 3a and shown as mean values ± SEM (n=3 independent experiments). *P<0.05;
**P<0.01; ***P<0.001, as per two-way ANOVA with Tukey’s post-hoc test.
Bottom: To determine the optimal dose of palmitate in mHypoA-NPY/GFP neurons, dose curve analysis was performed and
relative changes in Bax/Bcl2 mRNA were assessed. 50 µM palmitate significantly increased Bax/Bcl2 mRNA at 4 and 24 hours
following treatment (D). However, 25 µM palmitate did not significantly alter this ratio (C). Both 25 and 50 µM palmitate
significantly upregulated AgRP transcript levels at 24 hours (E), but only a 25 µM dose of palmitate was used for the remaining
studies. Data was normalized to histone 3a and shown as mean values ± SEM (n=3-4 independent experiments). *P<0.05;
**P<0.01; ***P<0.001, as per two-way ANOVA with Tukey’s post-hoc test.
A B
C D
E
41
exposed to 50 ng/mL TNF-α for up to 24 hours and gene expression of AgRP and certain
pro-inflammatory markers (IkBα, NF-κB and IL-6) was assessed using qRT-PCR. Analysis
of the results indicates that in the mHypoA-NPY/GFP neurons, AgRP gene expression was
significantly increased by TNF-α at 4, 8 and 24 hours following treatment (n=3; P<0.05; Fig.
3.2A). At 2 to 4 hours following treatment, TNF-α also upregulated mRNA levels of NF-κB
(n=3; P<0.05; Fig. 3.2B), IkBα (n=3; P<0.05; Fig. 3.2C), and IL-6 (n=3; P<0.05; Fig. 3.2D).
These findings demonstrate that TNF-α, like palmitate, can regulate the expression of AgRP
while inducing/exacerbating an inflammatory state in mHypoA-NPY/GFP neurons.
3.3 Palmitate-mediated regulation of AgRP gene expression is metabolism-dependent
Current literature has demonstrated the importance of fatty acid metabolism to overall energy
homeostasis. For example, DIO mice treated with myriocin (an inhibitor of de novo ceramide
synthesis) show a complete reversal of glucose intolerance and peripheral insulin resistance
(106). Moreover, central administration of inhibitors of β-oxidation reduce food intake (54).
However, whether fatty acid metabolism plays a role in palmitate-mediated regulation of
AgRP gene expression is unknown. Therefore, to determine whether palmitate-mediated
regulation of AgRP gene expression is palmitate metabolism-dependent, the mHypoA-
NPY/GFP neurons were treated with methyl palmitate. Methyl palmitate cannot be
metabolized, as the additional methyl group prevents activation of the fatty acid to palmitoyl-
CoA by the long chain fatty acyl-CoA synthetase enzyme. Treating our neurons with
different doses of methyl palmitate (25, 50 and 75 µM) for 24 hours did not result in similar
upregulation of AgRP mRNA as seen following 25 µM palmitate treatment (n=4; Fig. 3.3).
This result suggests that palmitate-mediated upregulation of AgRP gene expression is
palmitate metabolism-dependent.
42
Time (h)
Ag
RP
/His
ton
e m
RN
A
0 1 2 4 8 240.0
0.5
1.0
1.5
2.0
2.5H2O
50 ng/mL TNF-
*
****
*
2 40.0
0.5
1.0
1.5
Time (h)
NF
-kB
/His
ton
e m
RN
A
*
Time (h)
IkB
/His
ton
e m
RN
A
2 40.0
0.5
1.0
1.5
2.0*
2 40.0
0.5
1.0
1.5
Time (h)
IL-6
/His
ton
e m
RN
A
H2O
50 ng/mL TNF-*
A
B C
D
Fig. 3.2 TNF-α upregulates pro-inflammatory and Agrp gene expression.
Top: mHypoA-NPY/GFP neurons were treated with 50 ng/mL TNF-α (grey) or vehicle (black), RNA was isolated from 1 to 24
hours and analyzed using quantitative real-time PCR with a primer for AgRP (A). Treatment with 50 ng/mL TNF-α resulted in a
significant increase in AgRP transcript levels at 4, 8 and 24 hours. Data was normalized to histone 3a and shown as mean values
± SEM (n=3 independent experiments). *P<0.05; **P<0.01; ***P<0.001, as per two-way ANOVA with Tukey’s post-hoc test.
Bottom: mHypoA-NPY/GFP neurons were treated with 50 ng/mL TNF-α (grey) or vehicle (black), RNA was isolated from 1 to
4 hours and analyzed using quantitative real-time PCR with primers for NF-κB (B), IκBα (C) and IL-6 (D). Treatment with 50
ng/mL TNF-α resulted in a significant increase in NF-κB transcript levels at 4 hours, a significant increase in IκBα transcript
levels at 2 hours and a significant increase in IL-6 transcript levels at 4 hours. Data was normalized to histone 3a and shown as
mean values ± SEM (n=3 independent experiments). *P<0.05; **P<0.01; ***P<0.001, as per two-way ANOVA with Tukey’s
post-hoc test.
43
25 uM 50 uM 75 uM0.0
0.5
1.0
1.5
Treatment Dose
Ag
RP
/His
ton
e m
RN
A
H2O
Methyl Palmitate
mHypoA-NPY/GFP, AgRP, 24 hrs
Fig. 3.3 Methyl palmitate does not regulate Agrp gene expression.
mHypoA-NPY/GFP neurons were treated with 25, 50 or 75 µM methyl palmitate (grey) or vehicle (black), RNA was
isolated at 24 hours and analyzed using quantitative real-time PCR with a primer for AgRP (A). Methyl palmitate treatment
did not result in similar upregulation of AgRP mRNA as seen with 25 µM palmitate treatment. Data was normalized to
histone 3a and shown as mean values ± SEM (n=4 independent experiments). *P<0.05; **P<0.01; ***P<0.001, as per two-
way ANOVA with Tukey’s post-hoc test.
44
3.4 Palmitate triggers the phosphorylation of p38 MAPK in mHypoA-NPY/GFP
neurons
In peripheral tissues, palmitate has been shown to induce the phosphorylation and
activation of ERK1/2, JNK and p38 MAPK pathways. For example, palmitate induces
phosphorylation/activation of the MEK-ERK-IKK axis and pro-inflammatory cytokine
expression in skeletal muscle (107). In HepG2 cells, palmitate induces hepatic insulin
resistance through activation of JNK and p38 MAPK pathways (108). Yet, the direct effects
of palmitate on signaling events in specific hypothalamic neurons remain relatively
unknown. Recent work by Mayer et al. has shown that palmitate induces JNK
phosphorylation in a hypothalamic, neuronal cell model, mHypoE-44 (109). To test whether
any of the aforementioned pathways become activated in response to palmitate exposure in
mHypoA-NPY/GFP neurons, Western Blot analysis was used to analyze the phosphorylation
status of these key signaling kinases. Although palmitate did not induce phosphorylation of
ERK1/2 or JNK, it induced phosphorylation of p38 MAPK after 30 minutes of exposure
(n=3; P<0.05; Fig. 3.4A). This result may provide some insight into the mechanism by which
palmitate mediates its regulation of AgRP gene expression.
3.5 Palmitate pre-treatment dampens the mHypoA-NPY/GFP neurons’ response to
insulin
High-fat diets and elevated levels of saturated fatty acids have been shown to result in
impaired insulin signaling, which can be defined as a state of insulin resistance. Saturated
fatty acids are TLR4 agonists, and a study by Shi et al. demonstrated that TLR4 is essential
for acute lipid-induced insulin resistance (110). While many studies have begun to determine
45
Time (min)
Rela
tive p
38 M
AP
K P
ho
sp
ho
ryla
tio
n
5 15 30 600.0
0.5
1.0
1.5H2O
25 uM Palmitate
* mHypoA-NPY/GFP, 30 min
- +
p-
p38MAPK
Gbeta
A B
Fig. 3.4 Palmitate induces phosphorylation of p38 MAPK.
mHypoA-NPY/GFP neurons were treated with 25 µM palmitate (grey) or vehicle (black), and protein was harvested
after 5, 15, 30 or 60 minutes. Relative phospho-p38 MAPK was measured using Western Blot analysis and normalized
to Gβ, which served as a loading control. Results indicate that 25 µM palmitate significantly increased p38 MAPK
phosphorylation at 30 minutes (A). Representative blots are shown in B. Data are shown as mean values ± SEM (n=3
independent experiments). *P<0.05; **P<0.01; ***P<0.001, as per two-way ANOVA with Tukey’s post-hoc test.
46
the consequences of dyslipidemia in peripheral tissues, little is known about the effects of
excess lipids in the brain. Recently, Mayer et al. demonstrated that prolonged exposure to
palmitate impairs insulin activation in the mHypoE-44 hypothalamic neuronal cell model, as
assessed by phosphorylation of Akt (109). Whether palmitate has similar effects on insulin
signaling in representative NPY/AgRP neurons is unknown. To determine the effects of
prolonged palmitate exposure on neuronal insulin signaling, we pre-treated mHypoA-
NPY/GFP neurons with 25 µM palmitate for 24 hours. After palmitate pre-treatment, cells
were washed with PBS and subsequently serum-starved for one hour. Cells were then re-
challenged with 10 nM of insulin to determine neuronal responsiveness. Using Western Blot
analysis, it was determined that 24 hours of palmitate pre-treatment was sufficient to
significantly decrease insulin-mediated Akt phosphorylation in mHypoA-NPY/GFP neurons
(n=4; P<0.05; Fig. 3.5A).
Obesity is also associated with hyperinsulinemia (111). This increase in plasma
insulin levels is thought to be a contributing factor to the development and/or perpetuation of
insulin resistance. Therefore, to determine the effects of prolonged insulin exposure on
neuronal insulin signaling, we pre-treated mHypoA-NPY/GFP neurons with 100 nM insulin
for 24 hours. After insulin pre-treatment, cells were washed with PBS and subsequently
serum-starved for one hour. Cells were then re-challenged with 10 nM of insulin to
determine neuronal responsiveness. Using Western Blot analysis, it was determined that 24
hours of insulin pre-treatment was sufficient to significantly decrease insulin-mediated Akt
phosphorylation in mHypoA-NPY/GFP neurons (n=3; P<0.001; Fig. 3.5C). Collectively,
these data indicate that prolonged exposure to either palmitate or insulin impairs insulin
signaling in an NPY/AgRP neuronal cell model.
47
Time (min)
Time (min)
pAKT/AKT, 15 min, mHypoA-NPY/GFP
25 uM Palmitate Pre-Treatment
pA
KT
/AK
T (
A.U
.)
- +0
1
2
3
4PBS
10 nM Insulin
*
pAKT/AKT, 15 min, mHypoA-NPY/GFP
100 nM Insulin Pre-Treatment
pA
KT
/AK
T (
A.U
.)
- +0
1
2
3PBS
10 nM Insulin
***
pAKT –
AKT –
Pre-Treatment
mHypoA-NPY/GFP, 15 min
- + - +
pAKT –
AKT –
Pre-Treatment
mHypoA-NPY/GFP, 15 min
- + - +
B A
C D
Fig. 3.5 Prolonged palmitate or insulin exposure dampens the insulin-mediated increase in phospho-
Akt.
Top: mHypoA-NPY/GFP neurons were pre-treated with 25 µM palmitate (+) or vehicle (-) for 24 hours. Cells were
then washed and placed in serum-free media. Following 1 hour serum starvation, cells were re-challenged with 10
nM of insulin (grey) or vehicle (black) for 15 minutes. Relative phospho-Akt was measured using Western Blot
analysis and normalized to total Akt. Results indicate that 25 µM palmitate pre-exposure significantly decreased
insulin-mediated phosphorylation of Akt at 15 minutes (A). Representative blots are shown in B. Data are shown as
mean values ± SEM (n=4 independent experiments). *P<0.05; **P<0.01; ***P<0.001, as per two-way ANOVA with
Tukey’s post-hoc test.
Bottom: mHypoA-NPY/GFP neurons were pre-treated with 100 nM insulin (+) or vehicle (-) for 24 hours. Cells were
then washed and placed in serum-free media. Following 1 hour serum starvation, cells were re-challenged with 10
nM of insulin (grey) or vehicle (black) for 15 minutes. Relative phospho-Akt was measured using Western Blot
analysis and normalized to total Akt. Results indicate that 100 nM insulin pre-exposure significantly decreased
insulin-mediated phosphorylation of Akt at 15 minutes (C). Representative blots are shown in D. Data are shown as
mean values ± SEM (n=3 independent experiments). *P<0.05; **P<0.01; ***P<0.001, as per two-way ANOVA with
Tukey’s post-hoc test.
48
Chapter 4
Discussion
49
4. Discussion
4.1 General Discussion
Two functionally opposing neuronal populations in the arcuate nucleus of the
hypothalamus act as sensors for body energy stores and coordinate the activity of a complex
network of neurons that regulates feeding behavior and overall energy homeostasis. One
population expresses the orexigenic neuropeptides NPY and AgRP, while the other expresses
the anorexigenic POMC (α-MSH). These first-order neurons express receptors and have
intracellular molecular systems for detecting changes in the levels of hormones or nutrients
in the bloodstream (112). During fasting, the expression of NPY and AgRP are induced,
while POMC is inhibited. This coordinated response is in part the result of simultaneous
sensing of decreased nutrient availability and reduced levels of circulating insulin.
Conversely, in the postprandial state, NPY/AgRP neurons are inhibited and POMC neurons
are active. In this scenario, nutrient availability and insulin levels are increased. Therefore,
under normal physiological conditions, the hypothalamus acts under the control of peripheral
factors to maintain energy homeostasis (112).
When the balance is lost between food intake and energy expenditure, body adiposity
can no longer be maintained at a physiological level. This may lead to disturbances at the
hypothalamic level, which can cause metabolic disease. Excessive caloric intake is a primary
risk factor for the development of obesity, and a number of epidemiological studies have
shown that populations consuming diets high in fat are particularly prone to gaining weight
(55). According to Milanski et al., the long-chain saturated fatty acids are most harmful, and
can activate signal transduction through the toll-like receptor family leading to activation of
an inflammatory response in the hypothalamus (66). Another mechanism involved in the
50
breakdown of energy homeostasis is the development of resistance to the anorexigenic
effects of insulin (59). In studying the molecular mechanism(s) underlying this resistance,
many groups found that hypothalamic inflammation led to activation of intracellular
signaling pathways that promote negative cross-talk with insulin signaling pathways (113).
While classical in vivo approaches have enhanced our basic understanding of energy
homeostasis and highlighted the importance of NPY/AgRP and POMC neurons in the
maintenance of whole body energy homeostasis, such approaches cannot establish the direct
actions of nutrients (particularly fatty acids) on signaling events or neuropeptide gene
expression in specific hypothalamic neurons. Therefore, the purpose of this study was to
investigate the effects of saturated fatty acid palmitate on inflammatory state, neuropeptide
gene expression and signal transduction events in a representative NPY/AgRP cell model.
Here, the mHypoA-NPY/GFP cell line was used.
4.2 Plasma Non-Esterified Fatty Acids and the Determination of Palmitic Acid
Concentrations in the Brain
More than 95% of all fatty acids are stored as triacylglycerol fatty acids within
adipose tissue. Adipose tissue releases these stored fatty acids via lipolysis into the
circulation, where NEFAs become the major circulating lipid fuel (114). Lipolysis is a highly
regulated process and generally provides the exact amount of lipids to fat-oxidizing tissues.
For example, physiological increases in plasma insulin can reduce plasma NEFA
concentrations from their normal concentration of approximately 500 µM to 10-20 µM (115).
Conversely, under conditions of fasting, plasma NEFAs may increase to concentrations in
excess of 3000 µM (116). Excess fatty acid availability, however, can lead to increased
51
systemic NEFA release. This increase in systemic NEFA availability may contribute to the
adverse health consequences of obesity. Indeed, plasma concentrations of free fatty acids are
significantly elevated in obesity because 1) the enlarged adipose tissue mass releases more
FFA and 2) FFA clearance may be reduced (117). The saturated fatty acid palmitate (C16:0)
is one of the most pre-dominant NEFAs in the plasma of healthy individuals (118), and its
concentration nearly doubles in obese individuals.
In the brain, fatty acids serve both as an energy source and as components of
membrane lipids which maintain the structure and function of neuronal membranes. Fatty
acids are specifically sensed by hypothalamic neurons and, along with being involved in the
regulation of energy homeostasis, are required by the brain for regulation of hepatic glucose
production. Fatty acids cross the blood-brain barrier mainly by simple diffusion in the
unbound form, such that the concentration of fatty acids entering the brain is generally
proportional to their plasma concentration (119). However, longer chain fatty acids require a
facilitated uptake process to enter the cell. Fatty acid translocase (FAT/CD36) is one integral
membrane glycoprotein that has high affinity for long-chain fatty acids. This protein
mediates fatty acid dissociation from albumin and its “flip-flop” transport across the
phospholipid bilayer. Fatty acid binding proteins (FABPs) exist on the intracellular side of
the membrane, and provide additional means for the uptake of fatty acids.
In selecting a concentration of palmitate that could be used for treating the mHypoA-
NPY/GFP neurons, it was important to consider the physiological levels of palmitate that
would be seen by the brain. While little work has been done to determine exact
concentrations of palmitate in the brain, there are a handful of studies that enable us to make
a more accurate estimate. Given that the average total fatty acid concentration in the blood is
52
approximately 500 µM (115) and that palmitate comprises ~30% of total plasma NEFA
(152), we can say that the average concentration of palmitate circulating in the plasma is 150
µM. Determining how much circulating palmitate actually enters the brain required further
investigation of the literature. In 2000, Smith and Nagura calculated that the single-pass
extraction of radiolabelled palmitate from blood into brain was approximately 2% (64).
Recent work by Karmi et al. suggests that brain fatty acid uptake of radiolabelled palmitate
may even be increased in obese individuals compared to healthy controls (120). Combined
with the observation that plasma palmitate concentrations nearly double in obese individuals,
we can estimate that at least 6 µM of palmitate enters the brain. Accounting for an increased
uptake of palmitate in obese individuals, this number may be even higher. Therefore, the 25
µM dose of palmitate used to treat the mHypoA-NPY/GFP neurons reflects a roughly
physiological concentration that may be seen by the brain.
As the accumulation of lipids may lead to a lipotoxic response in the mHypoA-
NPY/GFP neurons, it was also important to consider the toxicity of the dose used.
Accumulation of lipids in non-adipose tissues (such as the brain) can lead to cellular
dysfunction and/or death. This may be the result of stimulating apoptotic pathways or
initiating cellular stress responses in the endoplasmic reticulum (ER stress). Mayer et al.
demonstrated the lipotoxic effects of palmitate on hypothalamic neurons, where doses greater
than 0.2 mM were sufficient to activate the MAPK JNK pathway, EIF2 (an ER stress
marker) and caspase-3 (a common cell death effector) in neuronal cells (109). In this thesis,
however, the Bax to Bcl-2 mRNA ratio was used to determine the neurons’ susceptibility to
apoptosis upon treatment with different doses of palmitate. Bcl-2 is a 26-kD protein that
blocks programmed cell death without affecting cellular proliferation, while Bax is a protein
53
that promotes apoptosis. Therefore, increases in the Bax to Bcl-2 ratio would indicate an
increase in susceptibility to apoptosis and vice-versa (121). Upon treatment with 50 µM
palmitate for 4 or 24 hours, there was a significant increase in the Bax to Bcl-2 ratio in
mHypoA-NPY/GFP neurons, suggesting an increase in susceptibility to apoptosis with this
dose. This assertion was confirmed by visual inspection using a light microscope, where
considerable cell death could be seen. However, treatment with 25 µM palmitate for 4 or 24
hours did not cause a significant change in the Bax to Bcl-2 ratio in mHypoA-NPY/GFP
neurons. This was one reason why the 25 µM palmitate dose was used for all subsequent
experiments. Interestingly, I could only treat my neuronal cell line with a maximum of 25
µM palmitate for 24 hours before cells began to die. In adipocytes, myocytes and β-cell lines,
investigators have used 500, 750 and 1000 µM doses of palmitate, respectively, for up to 48
hours (122-124). This implies that neurons may be more susceptible to the toxic nature of
saturated fatty acid palmitate.
4.3 Transcriptional Effects of Palmitate on mHypoA-NPY/GFP Neurons
While diet-induced obesity is associated with low-grade inflammation in peripheral
tissues, a similar process occurs in the hypothalamus. In 2005, evidence first emerged that
inflammatory changes could be detected in the brains of HFD-fed animals. In this study, 20
weeks of high-fat feeding increased reactive oxygen species and prostaglandin E2
production, along with upregulation of NF-κB signaling in the rat brain (58). In the
hypothalamus, De Souza et al. demonstrated that immune-related molecules (i.e. TNF-α, IL-
6) represent the largest class of genes with altered expression following an extended period
of high-fat feeding (59). Underlying this inflammatory response is the activation of both JNK
54
(59) and the classic IKKβ/NF-κB pathway, and the induction of endoplasmic reticulum stress
(66).
Recent research has focused on determining how HFD induces hypothalamic
inflammation. One potential mechanism is the ability of saturated, but not unsaturated, fatty
acids to activate toll-like receptor 4 (TLR4)/NF-κB signaling. Indeed, Milanski et al. found
that long-chain saturated fatty acids exert the most potent inflammatory stimulus within the
hypothalamus and that this inflammation is dependent on TLR4 activation (66). Palmitate, a
16-carbon saturated fatty acid, is capable of inducing NF-κB signaling through TLR4
activation both in neuronal cell culture and following direct infusion into the brain (65).
Therefore, we wanted to determine whether palmitate could elicit such an inflammatory
response in the mHypoA-NPY/GFP cell line. To do this, we assessed relative changes in
mRNA levels of NF-κB and IκBα following treatment with 25 µM palmitate using qRT-
PCR. Treatment with 25 µM palmitate was sufficient to increase NF-κB and IκBα mRNA
levels by 2 and 4 hours, respectively. However, it is unlikely that these changes in NF-κB
and IκBα mRNA were mediated at the transcriptional level. More likely, these changes were
due to changes in mRNA stability. Indeed, palmitate has been shown to activate miRNAs in
peripheral tissues (134), and these miRNAs may regulate mRNA transcript stability.
However, this cannot be confirmed until an RNA stability assay is performed (see future
directions). Nonetheless, these results suggest that the classic IKKβ/NF-κB inflammatory
signaling pathway is conserved in the mHypoA-NPY/GFP cell line and becomes activated in
response to the saturated fatty acid palmitate. This acute increase in pro-inflammatory
markers by palmitate may underlie the early-onset hypothalamic inflammation seen in diet-
induced obesity.
55
It is understood that fatty acids act on the central nervous system to modulate glucose
metabolism and overall energy homeostasis (52). For example, central administration of the
monounsaturated fatty acid oleate inhibits food intake and glucose production in rats (53).
These effects are accompanied by a decrease in expression of hypothalamic NPY after short-
term overfeeding, which suggests that small changes in plasma FA concentrations may serve
as a satiety signal to the CNS. Yet, little is known about the direct effects of fatty acids (such
as palmitate) on neuropeptide gene expression in specific hypothalamic neurons. Therefore,
we treated the mHypoA-NPY/GFP neurons with 25 µM palmitate over the course of 24
hours and assessed the regulation of AgRP mRNA levels using qRT-PCR. Treatment of
mHypoA-NPY/GFP neurons with 25 µM palmitate led to an increase in AgRP mRNA levels
at 24 hours. Unlike the changes in NF-κB and IκBα mRNA levels, the changes in AgRP
mRNA levels occurred after a longer time course. It is more likely, then, that the regulation
of AgRP mRNA levels by palmitate is mediated at the level of transcription. However, since
changes in mRNA levels may not accurately reflect changes in protein levels, AgRP protein
levels must be quantified in response to palmitate treatment before definitive conclusions can
be made. Regardless, this novel finding suggests that the saturated fatty acid palmitate
upregulates AgRP gene expression in the short-term, which may lead to increased feeding.
Indeed, this idea would fall in line with the observation that mice fed a high-fat diet tend to
have higher energy intake than normal-diet fed mice (125).
To begin to understand the mechanism(s) underlying palmitate’s acute regulation of
AgRP gene expression, we analyzed the AgRP gene promoter for potential transcription
factor (TF) binding sites using the online TF binding prediction tool Alibaba 2.1. The
program revealed potential binding sites for a host of transcription factors, including: NF-κB,
56
CREB and Elk-1. Given palmitate’s ability to activate the IKKβ/NF-κB inflammatory
signaling pathway and NF-κB gene expression in mHypoA-NPY/GFP neurons, it is
reasonable to believe that palmitate may modulate AgRP gene expression directly through
activation of NF-κB. While palmitate has not previously been shown to activate CREB in the
hypothalamus, it has been shown to phosphorylate and activate CREB in primary
hepatocytes (126). In the study by Collins et al., CREB phosphorylation by palmitate was
blocked by the inhibition of p38 MAPK. Indeed, p38 MAPK activation can lead to indirect
activation of CREB through MSK1 and thus, palmitate may modulate AgRP gene expression
through this pathway of CREB activation in hypothalamic NPY/AgRP neurons. Finally, Elk-
1 is a transcription factor that can become activated downstream of ERK1/2, JNK and/or p38
MAPK activation. Each of these MAP kinases has been shown to be activated by palmitate in
different peripheral tissues, making Elk-1 a candidate transcription factor of the AgRP gene.
For example, palmitate was shown to increase intramyocellular diacylglycerol (DAG) and
induce phosphorylation/activation of the MEK-ERK-IKK axis in skeletal muscle (107). In
HepG2 cells, palmitate-induced hepatic insulin resistance was found to be mediated through
JNK and p38 MAPK pathways (108). Though little is known about how palmitate modulates
intracellular signaling in the brain, it is plausible that palmitate may regulate AgRP gene
expression through activation of any of these MAP kinases.
4.4 Transcriptional Effects of TNF-α on mHypoA-NPY/GFP Neurons
As stated previously, De Souza et al. found that immune related molecules (i.e. TNF-
α and IL-6) were the largest class of genes with altered hypothalamic gene expression
following an extended period of high-fat feeding (59). Long-chain saturated fatty acids, such
as palmitate, appear to be the most potent inflammatory stimulus in the hypothalamus and
57
mediate their inflammatory effects via activation of the TLR4/NF-κB pathway. Downstream
targets of the transcription factor NF-κB include genes encoding for pro-inflammatory
cytokines, such as TNF-α. Cytokines (i.e. TNF-α) can then act as positive feedback signals to
enhance the inflammatory response by further activating inflammatory pathways through
receptors such as TNFR. Indeed, 50 ng/mL TNF-α significantly upregulated NF-κB, IκBα
and IL-6 mRNA levels within 4 hours of treatment in mHypoA-NPY/GFP neurons.
However, it is not known whether TNF-α (serving as a pro-inflammatory surrogate of
palmitate) can directly regulate AgRP gene expression. Therefore, mHypoA-NPY/GFP
neurons were treated with 50 ng/mL TNF-α for up to 24 hours and AgRP gene expression
was assessed using qRT-PCR. The results indicate that AgRP gene expression was
significantly increased by TNF-α at 4, 8 and 24 hours following treatment. Palmitate, being
able to upregulate NF-κB gene expression and its downstream targets (i.e. TNF-α), may thus
act to increase AgRP gene expression in mHypoA-NPY/GFP neurons by first upregulating
TNF-α gene expression.
4.5 Palmitate Metabolism and Signaling Dynamics in mHypoA-NPY/GFP neurons
While palmitate may indeed regulate AgRP gene expression through activation of the
TLR4/NF-κB pathway in mHypoA-NPY/GFP neurons, its effects may also be mediated by
its metabolites. The hypothalamus receives and processes nutrients that reflect the energy
status of the individual, and these “signals” can trigger the expression and secretion of
orexigenic and anorexigenic neuropeptides that regulate food intake and energy expenditure.
The idea that an intermediate of fatty acid metabolism could play a role in the regulation of
energy homeostasis has been supported by findings brought forth in previous studies. For
example, DIO mice treated with myriocin (an inhibitor of de novo ceramide synthesis) show
58
a complete reversal of glucose intolerance and peripheral insulin resistance (106).
Furthermore, hypothalamic CPT1 inhibition leads to increased intracellular levels of long-
chain fatty acyl CoAs, inhibition of feeding and reduced expression of ARC NPY and AgRP
(54). Yet, whether fatty acid metabolism plays a role in palmitate’s regulation of AgRP gene
expression is unknown. Therefore, to determine whether palmitate-mediated regulation of
AgRP gene expression is palmitate metabolism-dependent, mHypoA-NPY/GFP neurons
were treated with non-metabolizable methyl palmitate. Treating the mHypoA-NPY/GFP
neurons with different doses of methyl palmitate (25, 50 and 75 µM) did not result in a
similar upregulation in AgRP gene expression at 24 hours as seen following 25 µM palmitate
treatment. This finding suggests that palmitate-mediated regulation of AgRP gene expression
is palmitate metabolism-dependent.
Ceramide, a sphingolipid metabolite of palmitate, is one candidate mediator of
palmitate-mediated regulation of AgRP gene expression. A study by Ramirez et al. examined
the importance of ceramide synthesis to the orexigenic actions of ghrelin in terms of NPY
and AgRP gene expression. It was found that central inhibition of ceramide synthesis with
myriocin blocked the orexigenic actions of ghrelin and normalized levels of both NPY and
AgRP (127). Moreover, ceramide has been shown to activate NF-κB in human colon
epithelial HT-29 cells (135). This activation of NF-κB by ceramide may lead to upregulation
of TNF-α gene expression, which (as described in the previous section) may lead to changes
in AgRP gene expression. Nonetheless, it remains to be seen whether ceramides are involved
in palmitate-mediated regulation of AgRP gene expression. Future studies will involve the
treatment of mHypoA-NPY/GFP neurons with C16 ceramide to determine its direct effects
(if any) on AgRP gene expression.
59
In the periphery, palmitate has been shown to mediate its metabolic effects through
activation of ERK1/2, JNK and/or p38 MAPK. For example, Guo et al. demonstrated that
palmitate could induce apoptosis in both mouse 3T3-L1 and rat primary preadipocytes. In
this study, treatment of preadipocytes with palmitate induced ER stress responses and
increased the phosphorylation/activation of JNK and ERK1/2 (128). Similarly, palmitate
treatment induced ER stress through a JNK-dependent pathway in mHypoE-44 neurons
(109). In HepG2 cells, pamitate-induced hepatic insulin resistance was found to be mediated
through JNK and p38 MAPK pathways (108). Yet, it remains to be seen whether any of the
aforementioned pathways become activated by palmitate in the brain. Therefore, to assess the
signaling dynamics of palmitate in mHypoA-NPY/GFP neurons, we treated the cells with
palmitate for up to 1 hour and assessed the phosphorylation status of ERK1/2, JNK and p38
MAPK by Western Blot. The results indicate that palmitate increased phosphorylation of p38
MAPK, but not ERK1/2 or JNK. Closer examination reveals slight variations in control
(basal) levels of p38 MAPK over the course of this one hour time period, and this may be the
result of circadian clock-dependent oscillations in p38 MAPK activation. Indeed, Goldsmith
observed rhythmic p38 MAPK activation in cells derived from the suprachiasmatic nucleus
and fibroblasts of a mouse. However, in cells that lacked a functional circadian oscillator,
this rhythmic activation in p38 MAPK was lost and overall levels of p38 protein were lower
(137). Additionally, basal levels of p38 MAPK can change with movement. For this reason,
treatments were administered within the incubator in order to avoid excessive movement of
the plates.
Ultimately, the phosphorylation and activation of p38 MAPK is notable as the
transcription factor CREB can be indirectly activated downstream of p38 MAPK activation
60
(as described earlier). That palmitate induces phosphorylation of p38 MAPK only further
supports the notion that CREB may be a downstream mediator of palmitate’s regulation of
AgRP gene expression. Whether palmitate’s regulatory actions on AgRP gene expression
require p38 MAPK activation, however, remains to be elucidated (see future directions).
4.6 Palmitate Impairs Insulin Signaling in mHypoA-NPY/GFP Neurons
Central insulin action is critical for normal energy homeostasis. The orexigenic
NPY/AgRP and anorexigenic POMC neurons are responsive to insulin, and the coordinated
regulation of these two populations of neurons helps to maintain normal energy homeostasis.
However, many obese individuals develop insulin resistance and subsequent type 2 diabetes
mellitus. The excess circulating lipids found in obese individuals is a major contributing
factor to this insulin resistant state. One mechanism mediating this insulin resistance involves
serine phosphorylation and inhibition of insulin receptor substrate-1 (IRS-1). Saturated fatty
acid activation of IKKβ/NF-κB can lead to insulin resistance through this mechanism as
IKKβ becomes activated and serine phosphorylates IRS-1 (129). Serine phosphorylation of
IRS-1 can also occur through palmitate-induced activation of PKCθ (130). Moreover,
palmitate metabolites play a role, as ceramide can activate protein phosphatase 2A which
dephosphorylates pAKT and further contributes to this insulin resistant state (96). Therefore,
we wanted to assess the impact of palmitate on insulin signaling in the mHypoA-NPY/GFP
cell line. Our data suggest that prolonged palmitate pre-treatment (24 hours) significantly
dampens Akt phosphorylation in mHypoA-NPY/GFP neurons after 15 minutes of insulin re-
challenge. Indeed, the impairment of normal insulin signaling in these hypothalamic neurons
may have significant physiological impact, as it could lead to a dysregulation of neuropeptide
gene expression (AgRP, NPY) through impaired FoxO1 activity. Additionally, amino acids
61
were found to inhibit AgRP gene expression through an mTOR-dependent mechanism in the
GT1-7 hypothalamic cell line. By inhibiting mTOR signaling with rapamycin, cells exposed
to even high amino acid levels responded by increasing AgRP mRNA levels (138). Thus, it is
possible that dampening of Akt phosphorylation could also lead to a dampening of mTOR
activation. This may relieve the mTOR-dependent inhibition of AgRP gene expression,
contributing to further increases in AgRP gene expression.
Hyperinsulinemia is another hallmark feature of obesity. A study by Genuth found
that obese persons exhibited three fold increases in both fasting and postprandial insulin
levels (131). This increase in basal plasma insulin levels is thought to be a contributing factor
to the development or perpetuation of insulin resistance. It is likely, then, that continual
exposure of hypothalamic neurons to high levels of insulin may cause or contribute to the
development of an insulin resistant state. Therefore, we assessed the impact of prolonged
insulin exposure on insulin signaling in the mHypoA-NPY/GFP cell line. Our data suggest
that, like prolonged palmitate pre-treatment, lengthy insulin pre-exposure significantly
dampens Akt phosphorylation in mHypoA-NPY/GFP neurons.
4.8 Limitations
The hypothalamus consists of a multitude of fully differentiated neuronal cell types
regulating many bodily functions, including energy homeostasis. Hypothalamic neurons
express neuropeptides and may be controlled by both internal and external inputs. Due to the
complex nature of the hypothalamus, mechanistic studies of neuropeptide gene regulation
and elucidation of signaling events in these neurons is difficult to perform in vivo. Moreover,
classical in vivo approaches cannot be used to investigate the direct effects of a particular
62
agent on specific hypothalamic neuronal subtypes, largely due to the number of inputs
received from afferent neurons. Yet, cell lines immortalized from primary culture provide an
individualized model that lacks the complexity and integrated network of neuronal inputs
which makes studying these neurons difficult in vivo. Additional benefits of immortalized
cell lines include the fact that they can be maintained in a relatively controlled environment
with fewer uncontrolled variables, and different neuronal phenotypes can be used to
investigate the expression of specific genes or proteins (132). However, the use of
immortalized hypothalamic cell lines also has its limitations.
Although cell lines allow for a closer examination of the molecular processes at work
within a cell, the immortalizing gene SV40 T-antigen can interfere with these same cellular
processes. This may affect the pathways being studied. Initial studies conducted in our
laboratory using short-hairpin RNA knockdown of SV40 T-antigen demonstrate that T-
antigen expression can increase the basal activity of intracellular signaling kinases such as
Akt, Jak2 and STAT3 (Belsham, unpublished data). Therefore, I have attempted to reduce
the basal activity of such kinases using a combination of serum starvation and low glucose
during experimentation.
Another inherent limitation of this study is the physiological relevancy of the doses
used for treatments. In this thesis, the doses of TNF-α and palmitate used were chosen
primarily based on their degree of toxicity such that apoptosis was minimized in the
mHypoA-NPY/GFP neurons. Indeed, little research has been done to determine typical
concentrations of TNF-α or palmitate in the hypothalamus of obese versus healthy
individuals. While we can make estimates based on currently available literature regarding
the topic, it is still difficult to select physiologically (or pathologically) accurate doses for
63
treatments. Moreover, this study did not address the effects of other fatty acids (specifically
unsaturated fatty acids) on neuropeptide gene expression in mHypoA-NPY/GFP neurons.
Cintra et al. found that substitution of oleic acid (C18:1) into the diet of DIO mice was
sufficient to correct hypothalamic inflammation, hypothalamic insulin resistance and body
adiposity in these animals (136). How these fatty acids influence AgRP gene expression and
either potentiate or dampen palmitate’s effects on AgRP gene expression in mHypoA-
NPY/GFP neurons remain unknown.
Additionally, a long period of continuous passaging may change the cell
characteristics such that they become phenotypically different from the single cell population
of lower passage cells (132). Therefore, cell phenotype was continually monitored and a
maximum of 30 passages was performed. Furthermore, as with all in vitro studies, there are
limitations to their translatability to the in vivo setting. Immortalized neuronal cell models are
removed from the complex architecture and afferent cellular connections present in the intact
brain. Thus, it is challenging to account for the interneuronal communications within the
hypothalamus that are known to play important roles in feeding behavior and energy
homeostasis. For this reason, conclusions based on the results found here cannot be
generalized to all neurons or the hypothalamus itself.
While methyl palmitate was used to determine whether intracellular metabolism of
palmitate was required for palmitate-mediated regulation of AgRP gene expression, the
functional aspects of fatty acid methyl esters still remain largely unknown. Currently, it is
difficult to detect the localization of a specific fatty acid due to the lack of specific
radioactive or antibody labeling for a particular fatty acid chain. Thus, the only currently
available method of detecting the presence of fatty acid methyl esters is by using mass
64
spectrometry. However, this cannot be used to directly determine fatty acid localization
within specific tissue types. As such, whether methyl palmitate actually enters the cell cannot
be firmly established. Nonetheless, it is believed that the esterification of palmitate by a
methyl group in methyl palmitate prevents its activation by CoA. Lack of CoA activation
therefore abrogates any downstream metabolism of the fatty acid (150). Yet, recent literature
has suggested a possible anti-inflammatory role for methyl palmitate in different
experimental rat models (151). In light of these findings, the use of methyl palmitate may be
insufficient. Future studies will utilize another nonmetabolizable analog of palmitate, 2-
bromopalmitate, to further confirm that palmitate-mediated changes in AgRP gene
expression are palmitate metabolism-dependent.
Despite these limitations, the value of immortalized hypothalamic cell lines cannot be
underestimated. History demonstrates that cell models are reflective of natural physiology.
Importantly, the information gained from the study of these cell models will lead to a more
focused approach in the whole animal.
4.8 Future Directions
The thesis herein outlines the direct effects of saturated fatty acid palmitate on AgRP
gene expression. In addition, the experiments presented demonstrate palmitate’s ability to
induce a state of inflammation, phosphorylate/activate p38 MAPK and impair insulin
signaling in a hypothalamic neuronal cell model representative of NPY/AgRP neurons.
However, key questions remain. For one, whether palmitate-mediated changes in NF-κB and
IκBα mRNA levels are due to changes in mRNA stability remains unknown. Future studies
may employ an RNA stability assay to assess the stability of such mRNAs following
65
palmitate treatment. Moreover, the exact palmitate metabolite(s) that act to regulate AgRP
gene expression in mHypoA-NPY/GFP neurons have yet to be determined. Therefore, future
studies will utilize inhibitors for β-oxidation (etomoxir), protein palmitoylation (2-
bromopalmitate) and de novo ceramide synthesis (myriocin and/or fumonisin B1) to
determine which metabolite(s) may be mediating palmitate’s effects on AgRP gene
expression.
Additionally, while palmitate was found to induce the phosphorylation/activation of
p38 MAPK in mHypoA-NPY/GFP neurons, it was not determined whether this activation
was necessary for palmitate-mediated regulation of AgRP gene expression. Future studies
will therefore employ the use of a p38 inhibitor (such as SB239063) prior to palmitate
treatment to determine whether p38 MAPK activation is required for downstream regulation
of AgRP gene expression. In pursuing the idea that p38 MAPK activation may lead to
activation of the transcription factor CREB as a means of altering AgRP gene expression,
experiments will also be performed to: 1) determine the phosphorylation status of CREB
following palmitate treatment in mHypoA-NPY/GFP neurons and 2) utilize an inhibitor of
CREB prior to palmitate treatment to determine the effect of such inhibition on AgRP gene
expression.
Finally, the presence of functional GPR120 and adequate omega-3 fatty acids for its
activation is sufficient to lower systemic inflammation in mice (133). Likewise, genetic
disruption of GPR120 eliminates the ability of dietary omega-3 FAs to improve energy
homeostasis in obese mice (133). The anti-inflammatory properties of GPR120 activation by
omega-3 FAs have been demonstrated in adipocytes and macrophages, but little is known
about their actions in other areas of the body. Therefore, it would be of interest to examine
66
the effects of DHA pre-treatment on palmitate-mediated inflammation in mHypoA-
NPY/GFP neurons. Given the association between inflammation and insulin resistance,
additional studies will also be conducted to determine whether DHA pre-treatment can
prevent palmitate-mediated induction of insulin resistance.
4.9 Conclusion
The current thesis addresses the direct effects of the saturated fatty acid palmitate on
neuropeptide gene expression, signal transduction and insulin signaling in an immortalized,
hypothalamic cell model. Herein, palmitate has been shown to upregulate gene expression of
certain pro-inflammatory markers and AgRP in the mHypoA-NPY/GFP cell line. While
current literature has focused primarily on palmitate’s ability to activate TLR4/NF-κB
signaling, it was found that palmitate-mediated regulation of AgRP gene expression was
palmitate metabolism-dependent in the mHypoA-NPY/GFP neurons. Moreover, we provide
the first evidence that palmitate induces the phosphorylation/activation of p38 MAPK in a
distinct population of hypothalamic neurons. Finally, lengthy pre-exposure to palmitate or
insulin leads to impairment of normal insulin signaling in our cell model.
Physiologically, these findings may provide a molecular basis through which high fat
diets elicit their negative effects on feeding and overall energy homeostasis. Indeed, high fat
diet-induced obesity leads to increased feeding in mice over the long term, and this may be
due to continual upregulation of AgRP gene expression in NPY/AgRP neurons by saturated
fatty acids consumed in the diet. The finding that palmitate impairs insulin signaling in these
same neurons may only exacerbate the resultant increase in feeding. In beginning to elucidate
the signaling mechanisms through which palmitate acts in representative NPY/AgRP
67
neurons, we may also begin to identify potential therapeutic targets for the treatment of
obesity.
Taken together, these results add to our understanding of how fatty acids modulate
energy homeostasis within the hypothalamus. The knowledge gained through the analysis of
the mHypoA-NPY/GFP cell line will complement in vivo studies, and lead to a better
understanding of NPY/AgRP neuronal function in metabolic disorders such as obesity. This
study also indicates the importance of further neuronal cell line studies to better understand
the mechanism(s) underlying palmitate’s effects on neuropeptide gene expression and central
insulin signaling.
68
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