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IN VITRO EFFECTS OF OLEIC ACID ON THE
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HYPOTHALAMUS OF SPRAGUE DAWLEY AND DIET
INDUCED OBESE RATS -EXPLORING POSSIBLE
MECHANISMS

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IN VITRO EFFECTS OF OLEIC ACID ON THE RELEASE OF MONOAMINES
FROM THE HYPOTHALAMUS OF SPRAGUE DAWLEY AND DIET INDUCED
OBESE RATS -EXPLOR|NG POSSIBLE MECHANISMS

BY

Lakshmikripa Jagannathan

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Pathobiology

2010

Abstract

IN VITRO EFFECTS OF OLEIC ACID ON THE RELEASE OF MONOAMINES
FROM THE HYPOTHALAMUS OF SPRAGUE DAWLEY AND DIET INDUCED
OBESE RATS -EXPLORING POSSIBLE MECHANISMS

By
Lakshmikripa Jagannathan

One of the major health concerns that plague the modern world is obesity.
Significant proportions of a country’s health costs go towards the management of
obesity. Recently, understanding the changes that occur in the brain during
obesity is viewed as a potential source where the answers to major questions
around this disease lie. We were interested in exploring changes in the
neurotransmitters in response to fatty acids. Rat hypothalami were incubated in
an in vitro system, and changes in monoamine release in response to various
doses of oleic acid were measured by HPLC-EC. High dose of oleic acid
significantly increased the levels of norepinephrine (mean 115.008i22.927pg/mg
hypothalamus, p<0.0001), dopamine (39.495113.012pglmg hypothalamus,
p0.0011) and serotonin (mean 47.624t7.370pg/mg hypothalamus, p 0.0001)
efflux from the Sprague Dawley rat hypothalamus. This was blocked by the
addition of a PPAR alpha antagonist MK 886. In DIO rats there was release of
reduced response of norepinephrine and serotonin to high dose of oleic acid, and
the response was completely absent with dopamine. Thus free fatty acids could
act as potential signals stimulating neurotransmitter release in the hypothalamus
which possibly involves PPAR alpha. Differences between Sprague dawley and

BIG rats in fatty acid sensing may be a factor in the pathogenesis of this disease.

DEDICATION

"Gnanananda mayam dEvam N irmala spatikAkrudim!

Aadharam sarva vidhyanAm Hayagrlvam upAsmaheI!"

ACKNOWLEGEMENT

I owe my thesis to my mentor Dr P. S. MohanKumar and Dr Sheba
MohanKumar. They have been a source of unconditional support and
encouragement which has helped me immensely in successfully completing my
work. It would not be an exaggeration to say that this would have been an
impossible feat without their guidance. Every minute spent with the
MohanKumars has been a rich Ieaming experience and it has helped me grow
both as a student and as a person. Their positivity and benevolence will continue
to be an everlasting source of inspiration to me. I would like to thank my other
committee members Dr Tony Nunez and Dr loana Sonea for their highly
constructive suggestions on my work. Their encouragement has tremendously
boosted my confidence which has greatly helped in inching closer towards my
graduation. I would like to thank my lab mates Ebony, Madhan and Priya who are
also one of my closest friends, for helping me in every possible way both in and
outside the lab. Working with them has made my graduate study a memorable
and enjoyable experience. My lab manager Katrina Linning has been a fabulous
person to work with. She has contributed enormously towards increasing my
efficiency as a graduate student and her organizational skills are a huge source
of inspiration to me. I would like to thank my other friends Sarguru, Haritha,
Nandha, Ninitha, Madhu and Suga whose moral support helped me a long way in
finishing my writing effectively. I thank my counselor Christina for keeping meon
track. Last but definitely not the least; I thank. my family for being there for me

every step of the way.

TABLE OF CONTENTS

LIST OF FIGURES ......................................................................... vii
ABBREVIATIONS .......................................................................... ix
CHAPTER1
INTRODUCTION
I. Obesity
A. Obesity in the modern world ........................................................ 2
B. Modeling obesity in the lab animal ................................................ 3
C. Brain in the pathogenesis of obesity ............................................. 5
D. Neuroendocrine hypothalamus and obesity
i. Arcuate nucleus ................................................................ 6
ii. Paraventricular nucleus ..................................................... 7
iii. Ventromedial hypothalamus .............................................. 8
iv. Lateral hypothalamic area .................................................. 9
ll. Hypothalamus in the maintenance of energy homeostasis
A. Energy intake versus expenditure ............................................... 10
B. Peripheral signals
i. Hormonal signals to the hypothalamus ......................................... 10
ii. Nutrient signals to the hypothalamus ............................................ 11
C. Integration of various metabolic signals ............ ‘ ............................. 13
D. Efferent pathways
i. Autonomic pathways ........................................................ 14
ii. Behavioral pathways ......................................................... 15
iii. Endocrine pathways ......................................................... 15
Ill. Impaired hypothalamic signaling in obesity - Focus on free fatty acids
A. Impaired hypothalamic signaling in obesity .................................... 16
B. Free fatty acids and metabolism .................................................. 16
C. Types of free fatty acids ............................................................. 17
D. Hypothalamic sensing of free fatty acids ........................................ 17
E. Cellular mechanisms of fatty acid signaling .................................... 18
F. PPAR alpha ............................................................................. 19
G. Downstream pathways of fatty acid signaling ................................. 20
IV Regulation of hypothalamic neurons by catecholamines
A. Norepinephrine ......................................................................... 21
B. Dopamine ................................................................................ 21
C. Serotonin ................................................................................. 22
D. Potential regulatory role of monoamine sin feeding circuits ................ 23

CHAPTER 2
MATERIALS AND METHODS .............................................................. 26

CHAPTER 3
IN VITRO EFFECTS OF OLEIC ACID ON NEUROTRANSMITI'ER RELEASE
FROM THE RAT HYPOTHALAMUS - POSSIBLE MECHANISMS

I. Introduction ............................................................................. 32

II. Results ................................................................................... 33

Ill. Discussion .............................................................................. 47
CHAPTER 4

EFFECTS OF OLEIC ACID ON NEUROTRANSMI'ITER RELEASE FROM
HYPOTHALAMUS OF DIET INDUCED OBESE (DIO) MODEL OF RAT

I. Introduction .............................................................................. 53
II. Results .................................................................................... 56
III. Discussion ............................................................................. 68
CHAPTER 5
SUMMARY AND CONCLUSIONS ......................................................... 73
BIBLIOGRAPHY ................................................................................ 76

vi

LIST OF FIGURES

Figure 1-1 Hypothesis ....................................................................... 25

Figure 3-1 Effects of incubation of hypothalami with different doses of oleic acid
(DA) on Norepinephrine release ........................................................... 38

Figure 3-2 Effects of incubation of hypothalami with MK886, a PPARaantagonist
and 1.32 mM of oleic acid on Norepinephrine release .............................. 39

Figure 3-3 Effects of incubation of hypothalami with different doses of oleic acid
(GA) on Dopamine release .................................................................. 40

Figure 34 Effects of incubation of hypothalami with MK886, a PPARaantagonist
and 1.32 mM of oleic acid on Dopamine release .................................... ,.41

Figure 3-5 Effects of incubation of hypothalami with different doses of oleic acid
(GA) on Serotonin release ................................................................... 42

Figure 3-6 Effects of incubation of hypothalami with MK886, a PPARaantagonist
and 1.32 mM of oleic acid on serotonin release ....................................... 43

Figure 3-7. Effects of incubation of hypOthalami with different doses of oleic acid
(GA) on DOPAC content in the incubation medium .................................. 44

Figure 3-8 Effects of incubation of hypothalami with different doses of oleic acid
(GA) on 5HIAA content in the incubation medium ..................................... 45

Figure 3-9 Comparison of the dose response of monoamine neurotransmitters
released in vitro to different doses of oleic acid ........................................ 46

Figure 4-1 Body weight of Sprague Dawley versus DIO animals used in the in
vitro experiment ............................................................................... 59

Figure 4-2 Effects of high dose of oleic acid on Norepinephrine release from the
hypothalamus of diet induced obese rats ............................................... 60

Figure 4-3 Effects of high dose of oleic acid on Dopamine release from the
hypothalamus of diet induced obese rats ............................................... 61

Figure 4-4 Effects of high dose of oleic acid on Serotonin release from the
hypothalamus of diet induced obese rats ............................................... 62

Figure 4-5 Effects of high dose of oleic acid on DOPAC content in the incubation
medium following the incubation of hypothalami from diet induced obese
rats .............................................................................................. 63

vii

Figure 4-6 Effects of high dose of oleic acid on 5HIAA content in the incubation
medium following the incubation of hypothalami from diet induced obese
rats ................................................................................................ 64

Figure 4-7 Comparison of the norepinephrine response in vitro of hypothalami
from Sprague Dawley and DIO rats ...................................................... 65

Figure 4-8 Comparison of the Dopamine response in vitro of hypothalami from
Sprague Dawley and DIO rat ............................................................... 66

Figure 4-9 Comparison of the Serotonin response in vitro of hypothalami from
Sprague Dawley and DIO rat ............................................................... 67

viii

BMI
IMO
DR
NPY
AGRP
POMC
CART
TRH
CRH
ARC
VMH
LHA
PVN
GMSH
CCK
FATP
FABP
GPCR
PPAR
PPRE
CPT
ACS
FAS

ABBREVIATIONS

Body Mass Index

Diet induced obese

Dietary resistant

Neuropeptide Y

Agouti related protein
Proopiomelanocortin

Cocaine- and amphetamine-regulated transcript
Thyrotrophin releasing hormone
Corticotrophin releasing hormone
Arcuate nucleus

Ventromedial hypothalamus

Lateral hypothalamic area

Paraventricular hypothalamus
qulanocyte Stimulating Hormone
(fimbqwmmmn

Fatty acid transport protein

Fatty acid binding protein

G protein coupled receptor

Peroxisome proliferator activated receptor
Peroxisome proliferator activated receptor response element
Camitine palmitoyl transferase

Acetyl choline synthetase

Fatty acid synthase

EDTA
DHBA
KRH
OA

NE

DA
5HT
5HIAA
DOPAC

Ethylene diamine tetracetic acid
3,4-Dihydroxybenzylamine
Krebs Ringer Henseleit

Oleic acid

Norepinephrine

Dopamine

Serotonin

5 Hydoxyindoleacetic acid
3,4-dihydroxyphenylacetic acid

Chapter 1

Introduction

INTRODUCTION
I OBESITY
A. Obesity in the modern world

Obesity is one of the major health concerns in the modern world. Obesity
refers to a state of increased Body Mass Index (BMI) according to the National
Institute of Health definition. BMI for an individual is determined by dividing the
weight in kilograms by the square of the height of an individual in meters.
Although revised thoroughly over many decades, there is now consensus among
various national and international bodies on the definition of obesity‘. It is now
accepted universally that a BMI 2 25 is defined as overweight, and BMI 230 is
defined as clinically obese. Among obese individuals, the severity is further
categorized as class 1(BMI 30-349), classz (BMI 35-399) and classS
(BMI 240)‘. For children and adolescents, the BMI value is not fixed, however, a
BMI for age percentile distribution is used to determine whether the child is

obese or not 2.

Obesity has been growing in epidemic proportions in the United States
over the past three decades. According to the National Health and Nutrition
Examination Survey (NHANES) in the year 2007-2008, 33.8% of the adult
American population were obese and 68% were overweight“. The corresponding
prevalence of obesity for the year 1970-74 was only 14.1%2. A more disturbing
trend was observed with younger Americans where obesity prevalence was

11.9% in children aged between 2 and 19 years in the year 2007-2008‘.This is a

2

marked increase from a prevalence of 5% in the same age group during the

period from 1963-19705.

Obesity is seldom noticed as an independent entity and is often
associated with a multitude of other disorders which can be grouped together as
“metabolic syndrome”. Metabolic syndrome can be defined by the presence of
obesity concomitantly with dyslipidemia, type 2 diabetes, hypertension and a
proinflammatory state“. Obesity is also associated with risks for several diseases
including certain types of cancers’, arthritis", atherosclerosis” etc. Investigating
the mechanisms by which obesity is caused and how it leads to the precipitation
of other disorders will help us devise strategies to counter obesity and its

associated health risks.

Globally, it is estimated that-0.728% of a country’s healthcare expenses
go towards the management of obesity“. However, in the United States alone
the estimated cost for obesity remained at $113.9 billion in the year 2008, which
ranged from 5-1 0% of total healthcare spending during that year". Moreover,
other health effects associated with obesity such as cardiovascular disorders,
arthritis and cancer compound the economic effect. Thus, there is an urgent

need to address the problem of obesity.
8. Modeling obesity in the lab animal

Diet remains one of the foremost factors contributing to the current
prevalence of obesity worldwide. This is closely followed by lifestyle changes and

genetic factors. There have been many attempts to model the pathophysiological

3

changes that occur in human obesity. Several animal models of diet induced
obesity have been developed to study how genetics predisposes an animal to
develop obesity. In human beings, monogenic forms of obesity have been
identified, although they form only a small percentage of the various types of
obesity. More common forms of human obesity seen clinically are polygenic,
which also makes them inherently difficult to study through modeling‘2.The
monogenic forms of obesity as expected, have been easier to model, with classic
examples such as the ob/ob and db/db mice that lack leptin and leptin receptor
respectively“ ‘4. These animals are morbidly obese and show other features

reminiscent of metabolic syndrome and are popular in obesity research.

The polygenic form of obesity which is more commonly encountered in the
human population has also been modeled by dietary interventions using different
high-energy and high-fat diets“. Typically, common rodent strains like the
Sprague Dawley rat are fed obesogenic diets of varying composition and the
animals that develop a significant degree of obesity are categorized as diet-
induced-obese (DIO) and those which resist weight gain are categorized as
dietary-resistant (DR)‘6' 17. This type of obesity was taken to be polygenic as the
response to diet was normally distributed in the population. The extreme
phenotypes seen in these rodent colonies have been bred over many
generations to produce distinct DIO and DR strains“. While the former
monogenic animals are useful to delineate specific mechanisms of obesity, the
polygenic models are better suited for interventional studies as they closely

mimic the condition seen in the human population.

C. Brain in the pathogenesis of obesity

Obesity has been researched from the gastrointestinal, metabolic,
behavioral and hormonal standpoints. The greatest focus still remains on
metabolism, particularty in the intestines, liver, skeletal muscle and adipose
tissue‘9'2‘. Although theses peripheral organs play a crucial role in energy
homeostasis, it is the central nervous system that has emerged as a key player

in the etiology and pathogenesis of diet-induced obesity“ 22.

In the central nervous system, the hypothalamus is the primary center
which integrates peripheral signals to maintain energy homeostasis and
subsequently body weight. The hypothalamus maintains the balance between
energy intake and energy expenditure by at least two different mechanisms. One,
it senses the metabolic status and regulates feeding behavior through orexigenic
_ and anorexigenic peptides such as Neuropeptide (NPY), Agouti related protein
(AGRP), Proopiomelanocortin (POMC) and Cocaine- and amphetamine-
regulated transcript CART”. Secondly, it directly affects metabolism in the
periphery, through autonomic pathways, mainly at the level of the liver to

regulate glucose production, fat deposition and weight gain“.

Apart from these major mechanisms, numerous circuits that influence
feeding behavior are also involved like the endocannabinoid system, the
catecholaminergic systems, Thyrotrophin releasing hormone (TRH) and

Corticotrophin releasing hormone (CRH) 2527.

D. Neuroendocrine hypothalamus and obesity

The principal hypothalamic regions directly regulating feeding
behavior include the arcuate nucleus (ARC), the ventromedial hypothalamus
(VMH), the lateral hypothalamic area (LHA), and the paraventricular nucleus
(PVN) ”'32. It is of great interest to understand the regulatory mechanisms that

govern these neuronal subtypes and how this regulation is disrupted in obesity.
l. Arcuate nucleus

The arcuate nucleus is one of the most significant players in the
hypothalamus with respect to energy homeostasis. Being a circumventricular
nucleus, the arcuate nucleus is conveniently positioned outside the bounds of the
blood brain barrier; hence can easily access molecules from both the blood and
the cerebrospinal fluid”. Thus it makes sense that the neurons in the arcuate
nucleus act as the first relay point in the hypothalamic sensing of nutrient

molecules and hormones, and are termed as first order neurons“.

The arcuate nucleus has a high density of two major groups of first order
neurons involved in energy balance, the AGRP/NPY neurons and the
POMC/CART neurons“. AGRP and NPY are known as orexigenic peptides as
they increase feeding behavior while the POMC, CART, CRH and aMSH are
said to be anorexigenic since they inhibit feeding behavior“. A fasted state
activates the orexigenic AGRP/NPY group of neurons which in turn are inhibitory
to the anorexigenic POMC/CART neurons”. POMC is a precursor of or

Melanocyte stimulating hormone (GMSH) which acts on its receptor in various

6

brain regions and is inhibitory to feeding“. AGRP also acts as a direct
antagonist at the level of GMSH receptors”. The brain regions that express the a
MSH receptor include the PVN, VMH and LHA, which also contain second order
neurons“. Thus a balance between these two systems regulates feed intake and

maintains body weight in healthy individuals.
Paraventricular nucleus

The paraventricular nucleus has emerged recently as another important
centre in the regulation of feeding and energy metabolism. The paraventricular
nucleus is an important relay point in the sympathetic nervous system”. In
energy homeostasis, sympathetic innervations to the white as well as brown
adipose tissue play an important role in controlling energy expenditure and fat
mobilization from these organs°' 37' 38. Thus the paraventricular nucleus plays -a

direct role in the autonomic regulation of energy metabolism.

The paraventricular nucleus also contains CRH neurons which play a
significant role in regulating feeding behavior. CRH is known to be a direct

t39. There is also

inhibitor of feeding behavior and is a potent anorectic agen
anatomical evidence of close interaction between the CRH system and the
melanocortin system”. Above all, CRH is a central molecule in the hypothalamo-
pituitary-adrenal or the stress axis, and dysregulation of the stress axis is a

phenomenon seen to coexist with obesity“.

A third functional significance of the paraventricular nucleus lies in the fact

that it also contains Thyrotrophin releasing hormone neurons“. The thyroid axis

7

controls many aspects of energy metabolism including metabolism of nutrients,
thermogenesis, etc. Thus thyroid hormones also regulate body weight. To
summarize, the paraventricular nucleus is involved in energy homeostasis
through multiple pathways encompassing the sympathetic nervous system, the

hypothalamo-pituitary-adrenal axis and the hypothalamo-pituitary-thyroid axis.
Ventromedial hypothalamus

The ventromedial hypothalamus was once the most prominent nucleus of
the hypothalamus thought to be involved in energy homeostasis“. This was on
account of a number of reproducible experiments in which lesions in and around
the ventromedial hypothalamus produced obesity in laboratory rats“. However,
soon it was refuted with the hypothesis that obesity due to VMH lesions occurred
due to metabolic consequences of damage to autonomic neurons rather than
due to change in feeding behavior and the exact nature of the pathogenesis of
hypothalamic obesity was a matter of much debate“ ‘3. But recently, the
ventromedial hypothalamus has regained its role as a centre for regulation of
feeding following studies that demonstrated specialized glucose sensing
neurons“ as well as various neurons that respond to feeding related
neuropeptides“. Presently, there is little doubt that the ventromedial
hypothalamus has a significant role to play in modulating feeding and is probably

mostly inhibitory to feeding.

Lateral hypothalamic area

The lateral hypothalamic area is supposed to have an effect quite opposite
to the ventromedial hypothalamus, i.e., it mainly stimulates feeding behavior“.
The lateral hypothalamic area also contains glucose sensing neurons which are
predominantly inhibited by increases in extracellular glucose concentrations".
These neurons respond to reduced glucose levels in circulation with the
expression of the peptide orexin, which as the name indicates is orexigenic in

nature“.

Noththstanding the wealth of information gathered on how distinct areas
of the brain regulate feeding, better understanding of neuronal networks and
processes has lead to a shift in the focus from anatomical regions to functional
systems like the melanocortin system, dopaminergic, serotoninergic systems and
so on. More often than not, neurons belonging to feeding related circuits overlap
with more than one of the aforementioned nuclei and also extend into other

regions of the brain like the brain stem“.

ll HYPOTHALAMUS IN THE MAINTENANCE OF ENERGY HOMEOSTASIS
A. Energy intake versus energy expenditure

The balance between energy intake and energy expenditure determines
whether an animal is obese or not. This intricate balance is maintained by a
number of physiological systems of which the brain acts as the highest centre for
integration. Like any balancing system, the hypothalamus senses the metabolic
status of the animal from a variety of peripheral signals. These signals are then
integrated and appropriate efferent pathways are activated or humoral responses

are instituted to maintain equilibrium.
B. Peripheral Signals

The brain receives information on the nutrient status from a variety of
sources. These types of peripheral signals encompass hormones like insulin,
leptin, cholecystokinin (CCK) and ghrelin, nutrient signals such as glucose, fatty
acids and amino acids. Besides these, the brain also receives afferent inputs

from the autonomic nervous system from the gastrointestinal tract”.
I. Hormonal signals to the hypothalamus

Of all the metabolic signals that influence the activity of the
neuroendocrine feeding circuits, two major hormones have held the interest of
researchers for a long period of time, namely leptin and insulin 49"". Leptin is an
adipose tissue derived hormone whose gene, the ob locus was first isolated in

mice that carried mutations in this gene“ .Leptin is a pleiotrophic hormone which

10

acts mainly on the hypothalamus but also has receptors localized in many other
tissues like the lungs, liver, kidneys, hematopoietic cells and reproductive
organs”. In the hypothalamus, leptin exerts its effects on the hypothalamo-
pituitary adrenal axis53, hypothalamo-pituitary gonadal axis“ and the
hypothalamo-pituitary-thyroid axisss. Of great interest in energy homeostasis are
the effects that leptin has on the neuroendocrine neurons in the arcuate nucleus.
The arcuate nucleus as mentioned earlier, has a number of peptidergic neurons

that are critical for feeding behavior.

The second most important hormone regulating energy metabolism is
insulin“. Insulin is known to affect the melanocortin system in the
hypothalamus”. Insulin action in the hypothalamus is also known to affect

peripheral insulin sensitivity and glucose production”.

Other molecules that signal the hypothalamus include gut hormones such
as Cholecystokinin (CCK), glucagon like peptide (GLP-1) and ghrelin which act

as satiety signals and modulate feeding behavior 27.
ii. Nutrient signals to the hypothalamus

The hypothalamus also gets direct cues on the metabolic status of the
animal by actually sensing the nutrient molecules in the circulation“. Two
principal nutrient signals that regulate feed intake and body weight include

glucose and fatty acids“.

11

Glucose

Glucose sensing by the hypothalamus has been widely studied and is of
great significance in obesity and diabetes research”. Hypothalamic neurons,
particularly those in the arcuate nucleus are known to respond to extracellular
glucose concentrations by either excitation or inhibition“. Those groups of
neurons that respond by excitation are known as ‘glucose excited’ and those that
are inhibited are known as ‘glucose inhibited’ neurons. These two groups of
neurons have been characterized through electrophysiological and molecular
studies. One would expect that increase in extracellular glucose concentrations
would signal the brain in a way that feeding is inhibited, in other words the
orexigenic NPY neurons should be inhibited and the anorexigenic POMC
neurons should be excited61 .These differential effects may contribute to the
regulation of feeding behavior and subsequently body weight. However, evidence
to support this is still being gathered and much of the electrophysiological studies
in isolated neurons have produced uncertain results“. Glucose sensing at the
hypothalamus is also a key event in peripheral glucose homeostasis and is

impaired in type 2 diabetes and metabolic syndrome”.
Free fatty acids

While glucose signaling at the hypothalamus is only beginning to be
properly understood, information on fatty acid sensing by the hypothalamus is
even scarcer. The brain does not use free fatty acids as a source for energy but

at the same time, free fatty acids have been demonstrated to readily cross the

12

blood brain barrier and reach significant concentrations in the brain‘”.
Hypothalamic free fatty acid levels have been shown to change significantly with
feeding, but they are not a source of energy for the brain. This has raised the
interest in fatty acids as possible signaling molecules at the level of the brain
rather than a nutrient molecule“. Moreover, studies have shown that the same
groups of neurons that respond to glucose also respond to free fatty acids$5 and
extracellular glucose concentration was shown in another study to affect fatty
acid signaling in hypothalamic arcuate nucleus neurons“. Thus both fatty acids
and glucose appear to play a concerted role at the level of the hypothalamus in
the regulation of energy homeostasis. Impaired fatty acid signaling, just like
glucose and other hormone mediated signaling, can be explored as another
etiological factor in the development of diet induced obesity and the apparent

‘anergy’ of the hypothalamus to respond appropriately to metabolic signals.
B. Integration of various metabolic signals

It is now realized that the brain circuits that respond to metabolic cues and
influence feeding behavior and metabolism are highly complex, and the list of
molecules and regulatory pathways keep growing”. The major relay centers that
process this information include the hypothalamus and the brain stem“. A wide
variety of neuronal networks are involved in regulating feeding behavior”. The
most prominent ones are those in the hypothalamus, although numerous other
brain regions are also involved. Some other brain regions directly or indirectly
involved in modulating feeding related behavior include but are not restricted to

the brain stem“, the cortex and the limbic pathways”. The hypothalamic regions

13

controlling energy homeostasis have been discussed before (I D). Hypothalamic
neurons are known to project into the nucleus accumbens and ventral tegmental
area where reward calculation is made and motivation to eat arises". Also in
higher animals, the cortical centers are also involved in voluntary regulation of .
feeding and voluntary exercise behaviors". These regions integrate the

information on the metabolic status of the animal to recruit appropriate effectors.
C. Efferent pathways

Based on the metabolic signals the hypothalamus senses a fad versus
fasted state and recruits a variety of efferent pathways and also secretes various
effector substances that ultimately alter either feeding behavior or metabolism or

both.
i. Autonomic pathways

Autonomic projections from the brain and the spinal cord innervate
peripheral organs and control metabolism. Classical examples are the

sympathetic innervations to adipose tissue” 38

, vagal efferents to the liver and
gut”. While sympathetic innervations to adipose tissue may play a role in
lipolysis and lipid mobilization, vagal efferents to the liver and gut may promote

gluconeogenesis and increased fat deposition.

14

ll. Behavioral pathways

Behavioral pathways can be considered in two different ways. First is the
physiological feeding behavior that includes hunger and satiety. These behaviors
are vital to the survival of the organism and are direct responses to the metabolic
status. Specific areas of the hypothalamus and the brain stem regulate this kind
of feeding behavior”. The second type of behavior is the motivation to eat even
in the absence of the need or in some cases even when there is energy surplus
in the body. Brain circuits controlling reward and motivational behavior like the

mesollmbic dopaminergic system are involved in this kind of behavior 73.
iii. Endocrine pathways

Classical hormones such as cortisol, thyroxin, estrogen, testosterone, etc
control metabolism via established neuroendocrine axes. Examples include the
hypothalamo-pituitary-adrenal axis and the hypothalamo-pituitary-thyroid axis.
The target endocrine organ secretes the hormones in response to brain derived
releasing factors, and act on other visceral organs to regulate metabolism. A
number of other hormone-like substances are also secreted by peripheral organs
as a result of stimulation from the nervous system. Sometimes, the molecules
that act as signals also act as effectors. A good example of this is leptin, which is
shown to have a number of peripheral effects and central effects". Insulin

secretion is again contolled by nervous efferents.

15

III IMPAIRED HYPOTHALAMIC SIGNALING IN OBESITY - FOCUS ON FREE

FATTY ACIDS
A. Impaired hypothalamic signaling mechanisms in obesity

The complex nature of central nervous system feeding circuits gives way to
an equally complex variety of defects that could contribute to obesity. Thus it has
become extremely difficult to pin point a single mechanism in the pathogenesis of
obesity. However, defective signaling of the peripheral nutritional status to brain
regions regulating energy homeostasis has been a recurring motif in many forms
of obesity in laboratory rodents as well as humans”. Leptin signaling has been
widely studied and impaired sensitivity of the brain to leptin is seen as one of the
major events in the pathogenesis of obesity”. Free fatty acid is another
peripheral signal that could possible play a role in the pathogenesis of obesity in
the brain, however its role in the onset and development of obesity have not

been fully explored.
8. Free fatty acids and metabolism

The adipose tissue is a rich source of fatty acids in the complexed form of
triglycerides. Breakdown of triglycerides in the adipose tissue and other
peripheral organs contributes to the free fatty acids in circulation". Elevated free
fatty acid levels are noticed transiently in fasted states, and persistently in obesity
and diabetes. Elevated free fatty acid level seen in obesity is highly correlated
with insulin resistance and subsequent diabetes". A sustained elevation in free

fatty acids is seen in obese individuals with and without diabetes. Moreover these

15

individuals are more likely to develop insulin resistance later which in turn
contributes to increased lipolysis and an increase in circulating free fatty acids".
In laboratory animals, free fatty acids are found to be highly elevated in obese

animals, more so with high fat feeding (our unpublished data).
C. Types of free fatty acids

Biochemically, fatty acids are categorized based on the length of their
carbon chain and the presence of double bonds in their structure. Saturated fatty
acids are free of double bonds, and the common ones found in circulation include
palmitic acid, stearic acid and myristic acid. Unsaturated fatty acids contain one
or more double bonds. Monounsaturated fatty acids contain one double bond
and oleic acid is a classic example. Polyunsaturated fatty acids contain more
than two double bonds and include Iinoleic acid, Iinolenic acid and arachidonic
acid79. Various fatty acids have different physiological functions and hence are
also different in their intracellular signaling mechanisms. Oleic acid is commonly
studied as a representative fatty acid particularly as a nutrient signal” 8°' 8‘. Oleic
acid levels are one of the most prominently elevated following high fat feeding.
Impaired lipid metabolism and subsequent elevation in various plasma lipids

including free fatty acids, is a hallmark of obesity and metabolic syndrome“.
E. Hypothalamic sensing of fatty acids

Fatty acids have been demonstrated to readily traverse the blood brain
barrier 63. Free fatty acids are known to signal at the level of the brain particularly

the hypothalamus 65' 66. The elevated levels of free fatty acid seen in obesity most

17

probably alter the hypothalamic neuronal environment significantly to cause

various pathological changes seen in obesity.

Hypothalamic fatty acid sensing has been studied under physiological
concentrations both in vitro and in vivoaz‘“. Fatty acid levels have a profound
influence on feeding behavior and central administration of oleic acid was found
to inhibit feeding behavior“. It is also interesting to note that free fatty acid
signaling at the hypothalamus can have direct peripheral effects. One study
showed that central fatty acid signaling was important in the regulation of hepatic
glucose fluxes and glucose homeostasis”. Thus there appears to be
considerable evidence to support the role of central free fatty acids in energy

metabolism.

Neuronal responses to free fatty acids have also been demonstrated
directly. Isolated groups of neurons from the arcuate nucleus and the
ventromedial hypothalamus respond to extracellular free fatty acid levels“ 82.
Moreover, neurons important in the feeding circuitry like POMC neurons were
shown to be directly modulated by free fatty acids“. However, the mechanism of

fatty acid signaling in the hypothalamus has not been explored completely.
F. Cellular mechanisms of fatty acid signaling

Fatty acid signaling has been well characterized in the periphery.
Numerous protein acceptor molecules have been identified that can bind to free
fatty acids. At the level of the plasma membrane, fatty acid binding receptors

belonging to the G protein coupled receptor family have been identified of which

18

GPCR40 has gained attention”. Fatty acid transport proteins (FATPs) and fatty
acid binding proteins (FABPs) are also recognized as crucial to the
transmembrane signaling of free fatty acids”. But the most widely studied
molecules are the nuclear receptors belonging to the peroxisome proliferated
receptor (PPAR) group, mainly because of their direct effect on transcription of
various genes. The latter are a group of nuclear receptors that are further
classified into alpha, beta/delta and gamma subtypes. PPAR alpha, beta/delta
are expressed in a wide variety of tissue and their function is mainly regulation of
energy homeostasis. PPAR gamma is almost exclusively expressed in the
adipose tissue and is involved in adipocyte differentiation and other adipocyte

functions”.
G. PPAR alpha

Long chain fatty acids, including oleic acid, have been demonstrated to
activate PPAR alpha” 9‘. PPAR alpha expression has also been demonstrated
in the brain”. PPAR alpha is a transcriptional regulator of a number of genes
involved in lipid oxidation, fatty acid transport and many other metabolic
processes” 94. It plays a prominent role in the liver as well as the intestines94 in
the regulation of fat metabolism. Brain PPAR alpha activation is known to be
impaired when glucose levels are altered and also in PPAR knockout animals
that are subjected to fasting”. Thus there is a huge potential for PPAR alpha to

act as a signaling molecule for fatty acids in me brain.

19

H. Downstream pathways of fatty acid signaling

Free fatty acid binding molecules described above activate a variety of
other downstream molecules to bring about their physiological effects. At the
level of the pancreas, the Km: channels are important targets for free fatty acid-
induced regulation of insulin secretion. This channel has been demonstrated in
the central nervous system and possibly is a key player in the central regulation
of glucose metabolism“. For the PPARs, which are nuclear transcription factors,
the downstream targets are the regions of DNA that bind to these molecules and
regulate transcription. These regions are termed as PPRE or Peroxisome

Proliferator Activated Receptor Response Elements”.

The most widely known functions of PPAR alpha are similar to other
nuclear receptors. These receptors when activated by endogenous or exogenous
ligands heterodimerize with retinoid X receptor (RXR); this dimer subsequently
modulates transcription of specific genes via binding to PPREs. This is the
classical genomic effect of PPARsa9' 9". Some of the genes up regulated by
PPAR alpha activation include the Camitine palmitoyl transferase l and Il (CPT),
Acyl-CoA synthetase (ACS), Fatty acid binding protein (FABP), fatty acid
translocase (CD36) and mitochondrial Boxidation enzymes”. These proteins
ultimately lead to the uptake and utilization of free fatty acids by cells. Some of
the genes down regulated by PPAR alpha include Fatty acid synthase (FAS) and
acetyl-CoA carboxylase, which also leads to an overall decrease in free fatty
acids”. Thus PPAR alpha plays an important role in fat mobilization and
utilization.

20

IV REGULATION OF HYPOTHALAMIC NEURONS BY CATECHOLAMINES
A. Norepinephrine

Norepinephrine effects in the hypothalamus are most popularly associated with
two neuroendocrine systems, the hypothalamo-pituitary-adrenal axis and the
hypothalamo-pituitary-gonadal axis” 99. Noradrenergic cell bodies are mainly
located in the nucleus tractus solitaries (A1 /A2) and locus ceruleus (A6) regions
of the brain stem”. The effects of norepinephrine on hypothalamic neurons can
best be described as modulatory rather than directly stimulatory or inhibitory‘°°.
The stress axis and CRH are intricately connected with the neuronal networks
that regulate feeding behavior. Norepinephrine injection into the hypothalamus
has been demonstrated to modulate feeding behavior”. The effects of
norepinephrine on feeding have been stimulatory or inhibitory depending on the
site of injection as well as the type of receptor stimulated. Moreover,
norepinephrine also affects feeding behavior while interacting with other
neurotransmitters such as dopaminem. Thus norepinephrine exerts a significant
albeit complex effect on the neuroendocrine hypothalamus in the control of
feeding behavior. This is also reflected in the fact that many antiobesity drugs

also contain a norepinephrine reuptake inhibitor.
B. Dopamine

Dopamine is another significant neurotransmitter in the brain having a
wide range of localization and functions. The mesollmbic dopaminergic system

which is the centre for motivational behavior is of great interest in the study of the

21

behavioral changes leading to diet-induced obesity". Altered responses in this
region would explain why obese individuals are motivated to ingest highly
palatable foods irrespective of the satiety status of the body". Hypothalamic
dopamine is also an important player in feeding behavior. In a diet-induced
obese mouse model, high fat diet increased the expression of genes that
increased dopamine availability in the hypothalamus. Therefore, a sustained
reward effect to feeding might exist in these mice leading to over—consumption of
feed and increased weight gain1°3. Differential expression of dopamine receptor
levels were also seen in various brain regions, including the hypothalamus in
diet-induced obese mice‘“. Although not as well characterized as the
mesollmbic dopaminergic system, hypothalamic d0parnine also appears to play a

crucial role in regulating feeding.
C. Serotonin

Serotonin reuptake inhibitors are one of the most common types of
antiobesity drugs available. Serotonin thus possibly plays a crucial role in
regulating feeding behavior at the level of the brain. Serotonin agonists and
pharmacological compounds that increase the bioavailability of serotonin are
known to inhibit feeding behavior and reduce body weight ‘9. Specific subtypes of
serotonin receptors localized in the satiety related centers of the brain have been
implicated in the effect of serotonin on feeding behavior and energy balance”?
Serotonin has been shown to exert its effects in hypothalamic regions of the

PVN, VMH, suprachiasmatic nucleus and the dorsomedial nucleus“. Thus

22

changes in serotonin levels in the hypothalamus can substantially affect feeding

behavior.
D. Potential regulatory role of monoamines in feeding circuits

Monoamine neurotransmitters, which include both catecholamines,
(norepinephrine and dopamine) and indoleamines (serotonin) in the brain, have
pleiotrophic effects. The hypothalamus like the other brain systems is also a site
for the actions of these regulatory molecules. In general, norepinephrine and
serotonin in the brain induce satiety and therefore the drugs that inhibit their
reuptake have been a good choice for the treatment of obesity1°7. Dopamine is
slightly different in its effects in that it affects feeding behavior based on
motivation and reward characteristics‘”. In general, dopamine reuptake
inhibitors are also used to treat obesity. However, it may not be safe to
generalize their effects as their actions probably vary among various brain
regions and different physiological conditions. Monoamines have been
demonstrated to induce satiety in the perifomical region of the hypothalamus
which is also expected to be a downstream target for the first order neurons in

the arcuate nucleus‘“.
Hypothesis behind this study

Considering the significant role that monoamines play in the brain as a
whole and specifically the hypothalamus, we were interested in studying the
changes in monoamine neurotransmitters in the hypothalamus in response to

free fatty acids. We were also interested in understanding if there were significant

23

changes in this response in diet-induced obesity. In order to get a more complete
picture, we also wanted to explore the possible cellular mechanisms by which
free fatty acids could induce changes in neurotransmisslon. The brain PPAR
alpha appeared to be a good candidate for this purpose. PPAR alpha binds to
free fatty acids, particularly with high affinity to oleic acid. Moreover, PPAR alpha
has been demonstrated at the level of the hypothalamus to regulate feeding
behavior in concert with the fatty acid responsive enzymes”. One possible
mechanism by which these fatty acid responsive systems can affect feeding
behavior is through changes in monoamine neurotransmitters. Therefore, we
hypothesized that free fatty acids, oleic acid in particular can stimulate the
release of monoamines from the hypothalamus in vitro. We chose to use oleic
acid in our experiments because serum levels of oleic acid are significantly
elevated after feeding a high fat diet when compared to any other free fatty acid.
We also hypothesized that PPAR alpha plays a role in mediating the effect of

oleic acid on hypothalamic neurotransmitter release.

24

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25

Chapter 2

Materials and Methods

26

Animals

Adult male Sprague Dawley rats obtained from Harlan Sprague-Dawley,
Indianapolis, IN were used in experiment 1. Diet induced obese (DIO) were
purchased from Char1es River Laboratories, Wilmington, MA, USA. They were
bred in our colony. Male DIO rats were used in experiment 2 and were
compared with Sprague Dawley rats. All animals were housed in temperature
controlled (231:2°C) and light controlled (lights on from 0700 to 1900 hours)
environment and maintained on ad Iibitum water and regular rat chow. Animals
were used in the experiments in accordance with the National Institutes of
Health’s Guide for the Care and Use of Laboratory Animals and were approved
by the institutional animal care and use committee at MSU. Oleic acid complexed
with BSA was obtained from Sigma Aldrich (St. Louis, MO) and MK 886 (PPAR
alpha specific antagonist) was obtained from Cayman Chemicals (Ann Arbor,
MI).

On the day of the experiment, the animals were weighed and randomly
allocated to different treatment groups. The animals were sacrificed between
1000 to 1100 hours by rapid decapitation and their hypothalami were quickly
dissected out. The boundaries of the hypothalamus included the optic chiasm as
the anterior limit and the mamillary bodies as the posterior limit and the lateral

hypothalamic sulci as the lateral limits as previously described 1°9.

27

In vitro Incubation of hypothalamus

The hypothalamic tissue was weighed and incubated in a physiological
solution of Krebs’s Ringers Henseleit (KRH) in an in vitro incubation system as
described before mg. The hypothalamus was bisected sagitally into two halves
and incubated in a plastic tube containing 300 pl of KRH. The incubation
medium, KRH, consisted of 117m NaCl, 4.7 mM KCI, 1.2 mM of M9804, 1.2 mM
of KH2PO4, 2.5 mM of CaCl2, 24.8 mM NaHCO3, 11.1 mM of glucose; pH 7.4

which was maintained at 37 °C in a water bath. A mixture of 95% 02 and 5% C02

was bubbled through the tubes throughout the incubation. The incubation
medium was collected every hour for 5 consecutive periods of time. During the
first hour the medium was discarded as a prewash. The incubation medium from
the second hour was collected to measure the basal neurotransmitter release. At
the end of the second hour, the KRH solution was replaced with KRH containing
0.00132, 0.132 and 1.32 mM oleic acid, 50 pM MK 886 or 1.32mM oleic acid +
50 pM MK 886. The medium was then collected after one hour. The fourth
incubation was again done using KRH alone to look for residual effects of

treatment. During the final hour of incubation, the medium was replaced with high
K+ KRH to check the viability of the tissue. The media collected from various

incubations were stored with the addition of 15pl of 0.1 M perchloric acid at -70°C
until further analysis using High performance liquid chromatography with

electrochemical detection (HPLC-EC).

28

HPLC with Electrochemical Detection

The monoamines in the incubation medium were measured using HPLC-
EC method which has been described before 11°. The HPLC system consisted of
a LC-4C amperometric detector (Bioanalytical Systems, West Lafayette, IN,
USA), a phase II, 5 mm DS reverse phase, C-18 column (Phenomenex,
Tonance, CA, USA), a glassy carbon electrode, a CTO-10 ATNP column oven,
and an LC-10 AT VP pump (Shimadzu,Columbia, MD, USA). The composition of
the mobile phase was monochloroacetic acid (14.14glL), octane sulfonic acid
disodium salt (0.3 g/L), sodium hydroxide (4.67Sg/L), ultrapure EDTA (0.259IL) ,
acetonitrile (35ml/L) and Tetrahydrofuran (5mI/L) in nanopure water. The solution
was filtered through a 0.22pm filter through a Milli-Q purification system
((Millipore 00., Bedford, MA, USA) and degassed for 30 minutes. The mobile
phase was pumped through the HPLC system at a constant flow rate of 1.8
ml/min. The sensitivity of the electrode was 1 nA and applied potential was 0.65
V. The column oven was maintained at a temperature of 37°C in which both the
column and the working electrode were placed. Dihydroxybenzylamine (DHBA;
0.05M) was used as an internal standard for measuring the monoamines. At the
time of analysis, 30 pl of DHBA standard was added to 60 pl of the incubation
medium diluted with another 30 pl of 0.1 M perchloric acid. 100 pl of this mixture
was injected into the HPLC system by the autoinjector during each run.
Chromatograms were analyzed using the Chromatopac Class VP software
(version 7.2) (Shimadzu, Columbia, MD). Neurotransmitter levels were

expressed as pg/mg hypothalamus wet weight.

29

Statistical Analysis

To evaluate the viability of the hypothalamic tissue, it was first determined
using ANOVA if high potassium KRH mused a significant increase in
neurotransmitter release when compared to other incubations. The difference in
neurotransmitter release between different periods and also between different
treatment groups were analyzed using repeated measures ANOVA followed by

Fisher’s Least Significant Difference (LSD) test.

30

Chapter 3

In Wtro Effects of Oleic Acid on Neurotransmitter Release from the Rat
Hypothalamus - Possible Mechanisms

31

I. Introduction

An elevated level of circulating free fatty acids is a common feature of
obesity and the metabolic syndrome". These free fatty acids are known to cross
the blood brain barrier and exert their effects on brain regions such as the
hypothalamus“. It is of great interest to understand how high levels of circulating
free fatty acids affect neuronal circuits, particularly ones that regulate energy
metabolism. Oleic acid administered centrally has the potential to inhibit
feeding“. Thus under normal physiological conditions, high levels of circulating
lipids probably act as a signal to the brain to inhibit further feeding and utilize the
existing energy sources appropriately. However, when this homeostatic
mechanism is impaired it may contribute to pathologies like obesity and

metabolic syndrome.

The hypothalamus is the seat of major neuronal networks controlling
energy homeostasis”, although other brain regions are also involved in the
process. Free fatty acids have been demonstrated to have direct effects at the
level of the hypothalamus‘". Thus there appears to be necessary molecular
mechanisms in the hypothalamus to bind to circulating free fatty acids and sense
the changes in circulating free fatty acid levels. This is particularly interesting
because free fatty acids are not an important source of energy for the brain. Thus
apart from being an integral part of the cellular structures in the brain, free fatty

acids have a great potential to act as signaling molecules.

32

Oleic acid is one of the most highly elevated fatty acid in the circulation. It
is also one of the most highly abundant fatty acid in the human diet". The effects
of oleic acid on the hypothalamus have been studied to some extent 55' 83' 8" 8".
However, the effects of free fatty acids on monoamines have not been explored
at the level of the hypothalamus. Hence, we were interested in studying the
possible effects of various doses of oleic acid on norepinephrine, dopamine and

serotonin levels in the hypothalamus.

II. Results

Effect of different doses of oleic acid on Norepinephrine Release from the

rat hypothalamus in vitro

The effect of oleic acid on norepinephrine effiux from the hypothalamus is
shown in fig 3-1. There was a significant increase in norepinephrine release
during the 4th incubation period where hypothalami were exposed to high K+
KRH. This has a depolarizing effect on the hypothalamus and causes a
complete release of neurotransmitter release from the terminals. The marked
increase in norepinephrine release that was observed during the 4th incubation
period (p<0.0001) suggests that the hypothalami were viable throughout the

experiment.

There was no significant difference in norepinephrine release during the first

incubation period between hypothalami from different treatment groups. In

33

period 2, addition of 1.32 mM oleic acid stimulated norepinephrine release
(Mean1$.E; pg/mg hypothalamus) significantly into the incubation medium
(115008122927; p<0.0001) when compared to the control (871811.888),
0.00132mM Oleic acid (729611.685) and the 0.132mM oleic acid '
(38542119892) groups. There was a trend for norepinephrine release to
increase with higher doses of oleic acid however this was not statistically
significant. Incubation with high K+ KRH, produced a much lower release of
norepinephrine in the high dose oleic acid group (130624117245) compared to

the rest of the groups.

Effect of different doses of oleic acid on Dopamine Release from rat

hypothalamus

Dopamine release from the rat hypothalamus In response to different
doses of oleic acid is shown in fig 3-3. Dopamine release was increased
significantly after stimulation with high K+KRH indicating that the hypothalami
were viable during the previous 3 incubation. There was no change in dopamine
release between the different incubations in the control group. High dose of oleic
acid (1.32mM) produced a significant increase in dopamine release (pg/mg
hypothalamus; Mean1S.E) of 39495113012 during the second incubation which
was significantly higher than the control (526311.663; p<0.0011), low dose
(295111.796) and the medium dose (258711.019) of oleic acid. There were no

residual effects on dopamine release during the third incubation period.

34

Effect of different doses of oleic acid on Serotonin Release from the rat

hypothalamus

Serotonin release followed a pattern similar to the other monoamines and
increased significantly after high K+ stimulation when compared to the first 3
periods (p<0.001; Fig 3-5). During period 2 among various treatments, high dose
of oleic acid stimulated serotonin release (Mean1S.E; pglmg hypothalamus;
4762417370) when compared to the control group (p<0.0001). In this group
stimulation with high K+KRH produced only a modest increase in serotonin
release (1622319745) when compared with the control (p<0.0462). During
period 3 there was no significant change in serotonin release among different

groups.
I DOPAC and 5HIAA levels in response to various doses of oleic acid

Although stimulation with high K+ KRH produced a significant increase in

DOPAC release (Mean1S.E; pglmg hypothalamus) in all the treatment groups,
incubation with the high dose of oleic acid in the second period, lead to
significantly lower efflux of DOPAC into the incubation medium (47464112265)
when compared to the control group (199063159897; p<0.04). There were no
significant changes in the release of DOPAC with the other 2 doses of oleic acid

(fig 3.7).

5HIAA release appeared to be higher compared to DOPAC during all four

incubations (fig 3-8). During the second period, high dose of Oleic acid caused a

35

significant increase in 5HIAA release (Mean1S.E; pglmg hypothalamus;

10186521192929) when compared to the control group (306.668178747;
p<0.0013). During high K“ KRH stimulation, the release of 5HIAA into the

medium was highly variable between the different groups.

Effect of PPAR alpha antagonist on neurotransmitter release from rat

hypothalamus

To explore the possible mechanism of oleic acid-induced changes in
neurotransmitter release in vitro, the hypothalami were treated with the PPAR
alpha antagonist MK 886. Any effects of MK 886 by itself were studied by
incubating the hypothalamus with the antagonist alone. MK 886 alone did not
cause any change in the release of norepinephrine, dopamine, serotonin,
DOPAC or 5HIAA from the hypothalamus during the second incubation period. In
contrast, when the high dose of oleic acid was co-incubated with MK 886,
norepinephrine release was reduced to control levels (Fig 3-2). Norepinephrine
release (Mean1S.E; pglmg hypothalamus) during the second period with the
high dose of oléic acid was 115008122927 and this effect was blocked by co-

treatrnent with MK 886 (1323213207; p < 0.0001 ).

Dopamine release (Mean1S.E; pglmg hypothalamus) was also significantly
reduced (p<0.0045) by the combination of oleic acid and MK 886 treatment
(1986215623) when compared with high dose oleic acid alone (39495118012;
Fig 3-4). Addition of MK 886 to the medium brought down serotonin release
(Mean1$.E.; pglmg hypothalamus) following high dose of oleic acid from

36

4762417370 to 564812.066 (p <0.0001; Fig 3-6). There were no significant
changes in the release of 5-HIAA between the different treatment groups (Fig 3-
8). While high dose of Oleic acid significantly reduced DOPAC concentrations,
treatment with the combination of Oleic acid and MK886, prevented this

decrease, bringing oleic acid levels back up to that of the control group (Fig 3-7).

Fig 3-9 depicts the differences in the release of the three monoamines,
norepinephrine, dopamine and serotonin in response to incubation with the
different doses of oleic acid. The most dramatic changes were observed in
norepinephrine efflux compared to serotonin and dopamine. The effects of these
three neurotransmitters were completely blocked by incubation with MK 886, the

PPARqantagonist.

37

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lll Discussion

Among the monoamine neurotransmitters, the release of norepinephrine
from the isolated rat hypothalamus in vitro showed the greatest change in
response to oleic acid treatment. High dose of oleic acid produced a significant
increase in norepinephrine release when compared to controls group treated with
KRH alone. Although medium dose did not produce a significant change
compared to controls, there was an increasing trend from the low dose to the
high dose indicative of a dose response. Catecholamines injected into regions of
the hypothalamus including perifomical hypothalamus were found to be potent

suppressors of feed intake even in hungry ratsm'

. Interestingly, the same
catecholamines injected into the paraventricular nucleus of the hypothalamus
were shown to increase feed intake”. However, the well known anorectic agent,
amphetamine is a potent activator of catecholamine release” "4. Hence it is
possible that the overall effect of catecholamine release in the hypothalamus
would be in fact to reduce feed intake than increase it. In that context the,
increased norepinephrine release into seen following oleic acid possibly in a
dose dependent manner, could act as a homeostatic mechanism to indicate a
surplus of energy. This role of fatty acids has been demonstrated before 6" 8‘ and
fatty acids are considered as important signals for energy metabolism next to

glucose. However, fatty acid induced change in monoamine release in the brain

is a scarcely explored area.

47

With high K" KRH stimulation, there was a significant decrease in the

norepinephrine release from the hypothalamus that was treated with high dose of
oleic acid. This could be explained in the view of the neurotransmitter already
released during the previous incubation. The in vitro incubation system we used
provides for the mere maintenance of the tissue and cannot provide raw material

for biosynthesis of norepinephrine in this case.

It was interesting to note that responses in dopamine and serotonin at
high doses of oleic acid were similar to norepinephrine. Central dopamine is
another important regulator of feed intake"? The dopamine neurons extending
from the ventral tegmental area to the nucleus accumbens are the most widely
studied with respect to feeding behavior as they are established centers for
motivational behavior”. Dopamine deficient mice are aphagic and starve to
death in 4 weeks“. But interestingly, in these animals, restoration of dopamine
in the nigrostriatal dopaminergic system, rather than the mesolimbic

"6. Thus the exact mechanism of

dopaminergic system restored normal feeding
how dopamine regulates feeding is still not completely delineated. In the
hypothalamus, the most prominent dopaminergic system is the
tuberoinfundibular dopaminergic neurons that regulate prolactin secretion”.
However, dopamine in the hypothalamus is also known to be involved in multiple
other pathways including the feeding circuits”. Some studies clearly show that
dopamine signaling in the hypothalamus is necessary for regulation of feeding

and that this is impaired in obesity. Feeding was followed by a spontaneous

decrease in monoamine levels in the hypothalamus which was blunted in the

48

obese animals‘“. Our study shows a similar increase in dopamine and
furthermore it shows that these changes are in response to free fatty acids in

circulation.

Serotonin is another important monoamine neurotransmitter involved in
feeding behavior. It is generally thought of as a satiety promoting
neurotransmitter like norepinephrine and to some extent dopamine. Serotonin
levels in the hypothalamus like dopamine were also found to change
spontaneously following feeding which is a natural mechanism promoting
satiety“°. Depletion of serotonin centrally produced profound hyperphagia and
obesity in rats”. Conversely, the antiobesity drug sibutramine acts by enhancing
the availability of serotonin centrally, particularly at the level of the

120

hypothalamus . The increases in serotonin seen in this study may hence act as
a satiety signal in combination with norepinephrine and dopamine at the level of
the hypothalamus. This may further be involved in inhibiting feed intake.
However, live animal studies measuring feeding behavior must be performed to
ascertain the role of fatty acid induced increases in hypothalamic monoamines in

the control of feeding.

The metabolite of dopamine, DOPAC was significantly reduced in the
incubation medium following high dose of oleic acid. However, changes in
serotonin metabolite 5 HlAA did not show a consistent pattern worthy of
interpretation. This is in concordance with another study in which the
monoamines and their metabolites were measured from microdialysis in the

hypothalamus of rats to look at the spontaneous response to feeding 12‘. In this

49

study also, while dopamine increased with feeding, DOPAC levels reduced with
feeding. The converse was true for 5 HlAA responses, which also showed an
increased trend along with serotonin. Thus this pattern may be associated with
satiety at the level of the hypothalamus, and free fatty acids may play a

significant role in the whole process.

The release of all three monoamines was inhibited when high dose of
oleic acid was combined with a PPAR alpha specific antagonist MK886‘22. Free
fatty acids have been shown to act at the level of the hypothalamus through the
activation of PPAR alpha in regulating feeding behavior”. Oleic acid in particular
binds avidly to the PPAR alpha subtype of nuclear receptors than the other
PPARs‘”. Hence it is highly likely that oleic acid induced effects involve the
recruitment of the nuclear receptor PPAR alpha. However, there still remains a
huge gap in our understanding of how the nuclear receptor could possibly affect
neurotransmitter release. A number of possibilities can happen in this scenario.
The first thing to consider is the localization of the PPAR molecules in this scene.
PPAR alpha could be located in a neuronal site or even in other cells such as the
glial cells. PPAR alpha has been demonstrated in cultured neurons124 and also in
microglial cells‘zs. Thus PPAR alpha could directly act in the neurons
synthesizing the neurotransmitters or can act on glial cells which are also known
for their modulatory effect on the synaptic transmission in many regions of the

brain‘”.

Direct effects of PPAR alpha in an acute time frame as seen in this

experiment possibly involve rapid mechanisms apart from the conventional

50

nuclear signaling. Indeed, there have been studies where a rapid non-genomic
effect of PPAR alpha agonists have been noticed”. These have been mainly
through the direct modulation of ion channels to influence neuronal activity. A
similar modulation of monoamine release could be effected by PPAR alpha in the
terminals. The modulation of neurotransmitter at the terminal makes more sense
in this experiment as the effects were seen in isolated hypothalami. The cell
bodies of most of the monoamine neurons studied are located well outside the
hypothalamus. Whichever may be the mechanism of action, PPAR alpha in the
regulation of monoamine neurotransmission in the hypothalamus presents an

exciting new possibility in the field of obesity research.

Thus free fatty acids particularly oleic acid, can act at the level of the
hypothalamus directly to modulate serotonin, dopamine and norepinephrine
release in Sprague Dawley rats. This effect appears to be rapid and could
possibly be mediated by the intracellular fatty acid binding receptor PPAR alpha,
as specific antagonist to this receptor obliterated the response. Free fatty acid
induced elevation in the hypothalamic monoaminergic system could act as an
integral part of the complex events culminating in the maintenance of energy

homeostasis.

51

Chapter 4

Effects of oleic acid on neurotransmitter release from hypothalamus of Diet

induced obese (DIO) rat

52

I. Introduction

Obesity as seen in the modern world is mainly attributed to dietary factors.
Hence diet-induced obesity has been the most studied problem in metabolic
research. Diet-induced obesity also happens to be one of the most complex of all
metabolic pathologies and still continues to develop new unforeseen dimensions
giving opportunity for more research in this area. Diet-induced obese models in
laboratory animals have been extremely helpful in understanding the
pathophysiology of obesity. Initial models of diet-induced obesity were developed
in mice but later on, the rat models have gained popularity. Different methods
have been utilized to model obesity in rodents 1°. Single gene mutations
occurring spontaneously in genes that are crucial in maintaining body weight
have given rise to obesity models like the ob/ob, dbldb and fa/fa deficient
animals. The ob/ob is a leptin deficient mouse and the dbldb is a leptin receptor
deficient mouse model of obesity” ‘28. These have been used widely to study
the functions of the adipose tissue derived hormone, leptin 52. The faffa rat is also
known as the zucker rat, is deficient in a form of leptin receptor and shows many

features resembling metabolic syndrome in humans”.

Another type of laboratory animal model involves creating artificial
mutations in rodents to produce obesity which include irradiation-induced
changes as well as transgenic attempts to develop mutant mouse models“.
Again, such models are more useful to study functions of a single protein rather
than the evaluation of a complex pathology such as metabolic syndrome. The

model that is considered closest to mimicking human diet-induced obesity is the

53

polygenic model of obese rats developed from common laboratory strains such

as Wistar and Sprague Dawley rats".

A number of metabolic disturbances have been observed in these
models of obesity both centrally and peripherally. Diet-induced obese rats and
diet resistant rats differ in many metabolic features including feed intake, body
weight, thermogenesis, motor activity and insulin sensitivity“. The
pathophysiological bases for these are yet to be delineated clearly. There have
however been some important clues from some experiments done with these
animals. Obesity prone rats were found to have significant differences in their
monoamine turnovers particularly that of serotonin in the hypothalamus”?
Hypothalamic neurons from these animals characteristically have reduced leptin
sensitivitym. There was also a significant reduction in neuronal projections from
the arcuate nucleus to the PVN and also decreases in feeding-related peptide
neurotransmitters‘3‘. Although research into the possible mechanisms of
development of this phenotype has continued, the condition still remains poorly

understood.

These diet induced obese animals tend to gain more weight than control
animals when placed on a chow diet. A high fat diet produces a greater increase
in weight over and above that produced in response to a chow diet. The diet
resistant animals on the other hand fail to put on weight significantly even after a
high fat diet, and require highly concentrated forms of high fat diet to actually
make them gain significant amount of fat tissue”. The diet induced obese

animals demonstrate an increase in circulating leptin levels both when on a chow

54

diet as well as when they are fed with a high fat diet. They also develop insulin
resistance and have elevated circulating insulin levels. Other metabolic
abnormalities include increased blood glucose, plasma triglycerides, plasma free
fatty acids and cholesterol. They also showed had increased corticosterone

levels in the circulation and have a hyperactive stress axis 53.

Although the name implies that DlO rats become obese upon feeding,
changes in their neuronal responses have been demonstrated even before the

animals are fed on a high fat diet132

. Thus it is possible that anatomical and
molecular differences in the brain circuitry and subsequently variable brain
responses to nutrient molecules exist in obesity prone rats when compared to
normal rats. Hence we replicated the same conditions under which the Sprague
Dawley rat hypothalami were treated except that only the high dose of oleic acid
was used since it showed the maximal response with respect to change in
neurotransmitter release. We were interested in exploring the possibility of .

impaired fatty acid signaling at the level of hypothalamus as a potential factor in

the pathogenesis of diet induced obesity.

55

II. Results

Diet induced obese (DlO) rats were found to be significantly heavier than
the Sprague Dawley rats even on a chow diet. Body weight (MeantSE: 9) of
BIG rats was 632.2105 and was significantly higher than that of the Sprague
Dawley rats used in the experiment (479.4114]; p<0.05).

Effects of high dose of oleic acid on norepinephrine release from the DIO

rat hypothalamus in vitro

Effects of oleic acid on norepinephrine release from the BIO
hypothalamus are shown in Fig 4—2. As in the previous experiment, the
hypothalami were found to be viable at the end of the experiment by measuring
the release of norepinephrine following high K“ KRH. This was significantly
higher than any of the other incubations (p < 0.0001). High dose of oleic acid
(1.32mM) stimulated norepinephrine release (Meanis.E.; pglmg hypothalamus)
from the mo hypothalamus (4371513839) when compared with incubation with
KRH alone (2705011325). When the hypothalami were incubated with the
PPAR alpha antagonist MK 886 alone, norepinephrine release was
1218311494. When oleic acid was combined with the antagonist, the response
was inhibited significantly (2056715592; p<0.045). There were no residual

effects during the third incubation period. Norepinephrine release following high
K’ KRH stimulation was modest from hypothalami that were exposed to high

dose of oleic acid in the second incubation period.

56

Changes in norepinephrine release in both BIG and Sprague Dawley rats are
compared in Fig 4-7. While oleic acid produced a marked increase in
norepinephrine release in both groups, it was much attenuated in DIO animals

(p<0.05).

Effects of high dose of oleic acid on the release of Dopamine from DIO rat

hypothalamus in vitro

Effects of oleic acid on dopamine release from DlO rat hypothalami are
shown in fig 4-3. The DlO rat hypothalami were confirmed to be viable as there
was increased DA release (Mean1S.E; pglmg hypothalamus) following high
potassium KRH (p<0.0001). During period 2, there was no significant difference
between dopamine released from control hypothalami (1208016620) and
hypothalami treated with high dose of oleic acid (7 03512466). Similarly, there
was no change in dopamine release following treatment with MK 886 or a

combination of MK886 and oleic acid.

Fig 4—8 shows the differences in dopamine release between Sprague Dawley and
DlO rats. While oleic acid stimulated dopamine release from the hypothalami of
Sprague Dawley rats, it did not affect dopamine release from the hypothalami of

mo rats.

57

Effects of high dose of oleic acid on the release of Serotonin from DIO rat

hypothalamus in vitro

Fig 4-4 demonstrates the effects of oleic acid on serotonin release from
the hypothalami of DIO rats. As with norepinephrine, high dose of oleic acid
produced a significant increase in serotonin release (Mean1S.E; pglmg
hypothalamus) from the hypothalamus during the second incubation period
(21.135111.765) when compared to KRH treatment alone (1.6831673;
p<0.0295). While MK 886 alone did not produce any significant difference in
serotonin release (223711.324) when compared with control animals, the
combination of oleic acid and MK 886 increased serotonin release to 19.216.036.
These levels were comparable to levels stimulated by the high dose of oleic acid
and was significantly different from control animals (p<0.0287). There were no

residual effects of different treatments in the third incubation period.

Fig 4-9 shows the effects of oleic acid from the hypothalami of mo and Sprague
Dawley rats. While it increased serotonin release from the hypothalami of
Sprague Dawley rats, it did not have any effect on DlO rats. While the effect
observed in Sprague Dawley rats was completely blocked by MK 886, it

appeared to have no effect on DlO rats.

Effect of high dose of oleic acid on DOPAC and 5HIAA release from the

hypothalamus In vitro.

There were no significant changes in DOPAC and 5HIAA levels in

response to the different treatments in the second incubation period. Moreover,

58

 

the release of both DOPAC and 5HIAA remained higher compared to other

neurotransmitters and therefore, there was no significant increase in the release

of these metabolites after stimulation by high K+ KRH (Figs. 4-5 and 4-6).

59

Body Weight of Sprague Dawley and DIO rats

Body weight in grams

 

 

Sprague Dawley DIO

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significantly heavier than Sprague Dawley animals. *p<0.01

60

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65

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IV. Discussion

Diet induced obese model of rats that we used showed significant
changes in the pattern of neurotransmitter released when compared with
Sprague Dawley rats. This difference also varied across the different

monoamines that we measured namely norepinephrine, dopamine and serotonin.

High dose of oleic acid induced norepinephrine efflux which was
significantly greater than control groups. However, this change was still not up to
the extent of the norepinephrine release seen in the Sprague Dawley experiment.
(Fig 4-7). Moreover, the dose response that was observed in Sprague Dawley
rats was also absent in the DIO rats. Previous studies have shown that diet
induced obese rats have impaired monoamine transmission in the hypothalamus
12‘. Since monoamines act as important satiety signals, the obese animals
probably do not attain a state of satiety like Sprague Dawley animals and hence
tend to eat more leading to obesity. In our study we also show that this change is
brought about by free fatty acids, particularly oleic acid. Although a number of
lipid moieties including triglycerides and cholesterol circulate at high levels in the
plasma of DIO animals, it is the free fatty acid that is most readily able to cross
the blood brain barrier 63 and the hypothalamic centers regulating feed intake are
one of the first regions receiving this metabolic information”. Although the fatty
acids are readily available in DIO rats they may be unable to evoke a suitable
satiety response thereby leading to weight gain. MK 886 blocked this effect in
DIO rats also, showing the possible similarity between the two strains with

respect to the mechanism of norepinephrine release.

69

However, with dopamine, the changes observed in Sprague Dawley rats
were not seen in DIO rats. High dose of oleic acid completely failed to stimulate
dopamine release from DIO rat hypothalamus. In a study involving mice, the
opposite effect was noticed with high fat feedingm. In this study, high fat diet
produced an increase in genes increasing dopamine availability in the
hypothalamus. However, the changes observed in mice were in response to high
fat diet, while we examined the acute effect of fatty acid alone in DIO rats that
were still maintained on chow diet. These differences could have contributed to
the contradictory results. Dopamine at the level of the hypothalamus has a less
established role in feeding behavior as studies show that it can increase as well
as decrease feed intake depending on the region involved"? Since dopamine
reuptake inhibitors are also popular antiobesity drugs, it is safe to assume that
the overall effect of dopamine is to promote satiety. in that view, it appears that
free fatty acids fail to signal a state of satiety to the brain through the

hypothalamic dopamine in DIO animals.

Serotonin showed an even more peculiar pattem in the DIO rat model.
While the high dose of oleic acid continued to stimulate serotonin efflux from the
DIO rat hypothalamus, this effect was not abolished by the addition of PPAR
alpha antagonist. Although the release of serotonin was significantly higher when
compared to control DIO rats, comparison with Sprague Dawley rats showed that
this was still much lesser than that seen in the latter. The lack of response in
serotonin release with respect to MK 886 induced PPAR antagonism was an

unexpected finding. Diet induced obesity itself is associated with a number of

70

changes in the brain and specifically the hypothalamus of rats right from birth ‘3‘.
Changes in hypothalamic responses are seen in these animals even before they
are exposed to high fat dietm. Hence it is not unlikely that the DIO animals
recruit different mechanisms at the level of hypothalamus to regulate serotonin.
However, more studies on the mechanisms of serotoninergic transmission in the
hypothalamus are wananted before making any conclusions. Nevertheless, the
difference in serotonin release between Sprague Dawley and DIO animals
provides evidence that changes in serotoninergic transmission may be another
difference between these two animals and possibly an explanation to why these

animals are obese-prone.

Thus it appears that both norepinephrine and serotonin responses are
blunted in DIO animals. This in turn opens the possibility that the signaling
mechanisms leading to their release may be impaired. As MK 886 reversed the
norepinephrine release induced by high dose of oleic acid, PPAR alpha may
somehow be associated with noradrenergic neurotransmission in DIO rats. The
same cannot be said with certainty in the case of serotonin. Understanding the
exact link between fatty acids, PPAR alpha and monoamine release could
provide valuable insights into this phenomenon. Any of the downstream
molecules in this pathway could act as potential pharmacological targets in the

treatment of obesity.

A number of new drugs are available to treat obesity and many of them
act by increasing the monoamine neurotransmitter availability in the brain.

However, since they are not region specific, they affect all the monoaminergic

71

 

systems of the brain leading to unfavorable results"? If the exact mechanisms of
central nutrient sensing could be clearly delineated, drugs could be designed that
target particular molecules which are probably most associated with the
monoaminergic systems regulating feeding. This kind of specificity would be a

great boon to those undergoing treatment for obesity.

72

 

Chapter 5

Summary and Conclusions

73

 

Summary and Conclusions

Oleic acid at a high dose comparable to that seen in the circulation of
obese individuals was found to increase norepinephrine release from isolated rat
hypothalamus maintained under in vitro conditions. Although this response was
not significant with the medium and low doses, an increasing trend was noticed
with higher doses. This effect was completely blocked by the PPAR alpha
antagonist MK 886. Hence it is possible that free fatty acid sensing in the
hypothalamus involves PPAR alpha. This could possibly lead to a number of
molecular events culminating in the release of norepinephrine from the neuron.
Thus fatty acids could act as a potential signal at the level of hypothalamus to
induce satiety through the release of norepinephrine. The same response was
also noticed in DIO rat hypothalami subject to similar conditions. However the
release was much lower in DIO animals indicating that the satiety effect was less
pronounced in DIO rats. This may be a contributing factor in the hyperphagia

noticed in DIO rats and their subsequent tendency to gain weight.

Dopamine release was also increased following treatment of rat
hypothalami with a high dose of oleic acid. This effect was again blocked with the
addition of PPAR alpha antagonist. Hence in normal rats a high level of free fatty
acids can induce satiety by stimulating dopamine secretion. However, in contrast
to the noradrenergic response, dopamine stimulation was completely absent in
DIO rats. Thus DIO rats fail to produce enough dopamine at the level of the
hypothalamus to induce satiety. Although the effects of dopamine at the level of

the hypothalamus in controlling feeding behavior is still subject to controversy"5,

74

this difference seen between Sprague Dawley and DIO could have an important
relevance to the pathogenesis of diet induced obesity which may not be obvious

from the information from the current study.

Serotonin release was also stimulated by high dose of oleic acid in
Sprague Dawley rats and this effect was abolished by MK 886 in these animals.
However, in DIO rats although serotonin release was stimulated by a high dose
of oleic acid, PPAR alpha antagonism failed to reverse this effect. Hence it is
possible that different mechanisms controlling serotonin release may be
dominant in the DIO rat brain when compared with Sprague Dawley animals. The
amount of serotonin released was still lower in DIO rats than Sprague Dawley
rats. Hence, DIO animals still have reduced satiety recognition in response to

free fatty acids.

Thus free fatty acids can act at the level of hypothalamus to modulate the
release of various neurotransmitters involved in the regulation of feeding. Under
physiologiml conditions fatty acids could signal a fed state and inhibit further
feeding by stimulating the release of monoamines from nerve terminals in the
hypothalamus. This may be crucial mechanism in the regulation of feed intake
and body weight in a normal animal. Any disturbance in this intricate balance
could contribute to abnormalities such as obesity and metabolic syndrome.
Understanding the cellular mechanisms of feed intake regulation would help in
developing pharmacological agents to specifically target the central regions
regulating feed intake. Such specific pharmacological agents have the potential

to be safer and more efficacious anti obesity drugs.

75

 

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