The Pennsylvania State University

The Graduate School

Eberly College of Science

REGULATION OF NEUROPEPTIDE Y EXPRESSION AND THE SYMPATHO-

ADRENAL RESPONSE TO STRESS

A Dissertation in

Cell and Developmental Biology

by

Qian Wang

2011 Qian Wang

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Doctor of Philosophy

August 2011

The dissertation of Qian Wang was reviewed and approved* by the following:

Douglas Cavener

Professor of Biology

Co-Chair of Committee

Dissertation Advisor

Matthew Whim

Associate Professor of Cell Biology and Anatomy

Louisiana State University Health Science Center

Special member

Co-Chair of Committee

Bernhard Luscher

Professor of Biology, Biochemistry & Molecular Biology, and Psychiatry

Melissa Rolls

Assistant Professor of Biochemistry & Molecular Biology

Byron Jones

Professor of Biobehavioral Health

Zhi-chun Lai

Professor of Biology and Biochemistry & Molecular Biology

Head of Department of Cell and Developmental Biology

*Signatures are on file in the Graduate School

iii

ABSTRACT

Neuropeptide Y (NPY) is a 36 amino acid peptide that is synthesized by many neurons in

the central and peripheral nervous systems. NPY has been implicated in many physiological

functions including the regulation of feeding, anxiety, the stress response and cardiovascular

modulation. A single nucleotide polymorphism (SNP) in the NPY gene has been found to be

associated with elevated serum cholesterol levels, cardiovascular disease and diabetes. This SNP

changes the seventh amino acid in the signal sequence of the NPY preprohormone (preproNPY)

from leucine to proline. It is predicted to alter NPY synthesis, trafficking, or secretion. I tested

this hypothesis by expressing the mutant and wild-type preprohormones tagged with fluorescent

proteins in AtT-20 cells, an endocrine cell line. Prohormone from the mutant preproNPY was

synthesized and processed normally and entered the regulated secretory pathway. When

expressed in endocrine cells, NPY derived from both wild-type and mutant preproNPY were

found in the same large dense core granules. However the SNP altered the degree of co-

packaging and individual granules contained more NPY generated from the mutant preproNPY. I

conclude that the SNP enhances the packaging of NPY. This SNP also led to an increase in the

secretion of NPY, which is likely the consequential effect of the increased biosynthesis of the

prohormone. These findings are consistent with the observation that humans carrying this SNP

have higher levels of plasma NPY after exercise.

Peripheral NPY is derived from the sympathetic nervous system, which includes the

adrenal medulla. This tissue contains modified post-ganglionic neurons and is both part of the

sympathetic system and an endocrine gland. Mammalian adrenal glands consist of two regions,

the cortex and the medulla. Stress activates the HPA axis leading to a secretion of the

glucocorticoids (CORT) from the adrenal cortex. The sympatho-adrenal system is also activated

by stress leading to a release of catecholamines from the adrenal medulla. NPY and tyrosine

iv

hydroxylase (TH, a marker of catecholamine synthesis) are co-expressed in all mouse chromaffin

cells. Adrenal NPY transcription is known to be increased by various stressors and here I

investigate whether NPY has a regulatory role on adrenal activity. To address this question, mice

were exposed to an acute stressor the cold water forced swim test (cold FST) for 5-6 minutes. The

cold FST elevated both the electrical activity of chromaffin cells and the HPA axis. Unexpectedly,

activation of both limbs of the stress response was sustained even 24 hours after the cold FST.

The level of adrenal NPY-ir was significantly increased 24 hours after the cold FST both in vitro

and in situ, and this increase reversed one week later. The stress-induced increase in the levels of

adrenal NPY involved an up-regulation in the activity of the NPY promoter. To determine

whether NPY modulated adrenal activity, both wild type and NPY knockout mice were exposed

to the cold FST. Loss of NPY did not affect the stress-induced increase in plasma Cort, but the

removal of NPY significantly altered the neuronal branch of the stress response. That is, stress

increased the levels of adrenal TH-ir in situ in the wild type mice but the level of adrenal TH-ir

was not altered in the NPY knockout mice. Injection of BIBP 3226, a NPY Y1 antagonist was

associated with an increase in the level of TH-ir in adrenal slices in wild type mice indicating that

NPY negatively regulated adrenal TH via Y1 receptors. I conclude that acute stress has a long-

term effect on the synthetic capacity of the adrenal glands and that NPY plays an important role

in inhibiting the neuronal branch of the stress response.

v

Table of Contents

List of Figures ........................................................................................................................... ix

List of Tables ........................................................................................................................... .xi

Acknowledgements .................................................................................................................. xii

Chapter 1 Introduction ............................................................................................................ 1

1.1 Neurotransmitters in the nervous system ................................................................... 1 1.2 Hypocretin: one example of a prototypical neuropeptide .......................................... 2 1.3 Distribution of neuropeptide Y in the nervous system ............................................... 3 1.4 Processing and biosynthesis of neuropeptide Y ......................................................... 4 1.5 Physiological functions of neuropeptide Y ................................................................ 7

1.5.1 NPY in the cardiovascular system................................................................... 7 1.5.2 NPY in feeding and metabolism ..................................................................... 9 1.5.3 NPY in emotional regulation ........................................................................... 10 1.5.4 NPY in epilepsy .............................................................................................. 11

1.6 My thesis research on neuropeptide Y ....................................................................... 11

Chapter 2 Materials and Methods ........................................................................................... 14

2.1 Specific Methods for Chapter 3 ................................................................................. 14 2.1.1 Site-directed mutagenesis ................................................................................ 14 2.1.2 AtT-20 cells culturing ..................................................................................... 15 2.1.3 Transfection of AtT-20 cells ........................................................................... 15 2.1.4 Immunoprecipiation and Western blot ............................................................ 15 2.1.5 Immunocytochemistry ..................................................................................... 16 2.1.6 Image analysis ................................................................................................. 17

2.2 General Methods for Chapter 4 and Chapter 5 .......................................................... 18 2.2.1 Animals ........................................................................................................... 18 2.2.2 Cold water forced swim test ............................................................................ 18 2.2.3 Chromaffin cell cultures .................................................................................. 19 2.2.4 Immunocytochemistry ..................................................................................... 20 2.2.5 Cryostat frozen sectioning ............................................................................... 20 2.2.6 Immunohistochemistry .................................................................................... 21 2.2.7 Image analysis ................................................................................................. 21 2.2.8 Statistical analysis ........................................................................................... 22 2.2.9 Corticosterone ELISA ..................................................................................... 22

2.3 Specific Methods for chapter 4 .................................................................................. 23 2.3.1 Fox urine exposure .......................................................................................... 23 2.3.2 Semi-quantitative RT-PCR of NPY ................................................................ 23 2.3.3 RT-PCR of PP and PYY ................................................................................. 23

2.4 Specific Methods for chapter 5 .................................................................................. 24 2.4.1 Catecholamine ELISA ..................................................................................... 24 2.4.2 Antagonist injection ........................................................................................ 25 2.4.3 RT-PCR ........................................................................................................... 25

vi

Chapter 3 A Single Nucleotide Polymorphism Alters the Synthesis and Secretion of

Neuropeptide Y ................................................................................................................ 27

3.1 Introduction ................................................................................................................ 27 3.2 Results ........................................................................................................................ 30

3.2.1 Generation of the wild type and mutant NPY fusion protein constructs ......... 30 3.2.2 Expression of NPY prohormones derived from wild type and mutant

preproNPY........................................................................................................ 30 3.2.3 NPY prohormones derived from L7P and wild type preproNPY were

sorted similarly in AtT-20 cells ........................................................................ 31 3.2.4 NPY prohormones derived from L7P and wild type preproNPY entered

the same dense core granules ........................................................................... 32 3.2.5 Prohormones derived from L7P and wild type preproNPY were

differentially co-packaged ................................................................................ 33 3.2.6 NPY prohormones derived from L7P and wild type preproNPY were

sorted similarly in hippocampal neurons .......................................................... 34 3.2.7 Expression of NPY prohormone derived from mutant preproNPY leads to

increased NPY secretion................................................................................... 34 3.3 Discussion .................................................................................................................. 52

3.3.1 The SNP might affect the ER trafficking efficiency of the NPY

preprohormone ................................................................................................. 52 3.3.2 The SNP might cause a more efficient sorting of the NPY prohormone ........ 53 3.3.3 Codon usage does not explain the effect of the SNP ....................................... 54 3.3.4 Comparison of NPY T1128 with SNPs found in other peptide

preprohormones and prohormones ................................................................... 54 3.4 Conclusion ................................................................................................................. 55

Chapter 4 Acute Stress Increases the Levels of Adrenal Neuropeptide Y .............................. 56

4.1 Introduction ................................................................................................................ 56 4.2 Results ........................................................................................................................ 59

4.2.1 The cold FST activated the HPA axis and induced activity in chromaffin

cells ................................................................................................................... 59 4.2.2 GFP is expressed in the hypothalamus, hippocampus and adrenal medulla

in NPY (GFP) transgenic mice ......................................................................... 60 4.2.3 An NPY antibody specifically recognizes NPY in chromaffin cells............... 60 4.2.4 The cold FST led to an up-regulation of NPY-immunoreactivity in

chromaffin cells in vitro ................................................................................... 61 4.2.5 Brief exposure to fox urine also led to an up-regulation of NPY-

immunoreactivity in chromaffin cells in vitro .................................................. 62 4.2.6 The cold FST increased NPY-ir measured in adrenal slices ........................... 62 4.2.7 The cold FST-induced increase of NPY-ir in adrenal slices returned to

baseline one week after stress ........................................................................... 63 4.2.8 The cold FST increased GFP expression in NPY (GFP) transgenic mice ...... 63 4.2.9 Adrenal NPY mRNA expression was increased 3 hours after the cold FST .. 64 4.2.10 Pancreatic polypeptide and peptide YY mRNA expression in the adrenal

medulla after the cold FST ............................................................................... 65 4.3 Discussion .................................................................................................................. 89

4.3.1 Stress increases adrenal NPY transcription and peptide synthesis. ................. 89

vii

4.3.2 The mechanism that is involved in the stress-induced increase of adrenal

NPY. ................................................................................................................. 90 4.3.3 The transcriptional factors that are involved in response of the adrenal

medulla to stress. .............................................................................................. 90 4.3.4 Adrenal epinephrine in the stress response. .................................................... 92 4.3.5 Stress-induced secretion and synthesis coupling ............................................. 93

4.4 Conclusion ................................................................................................................. 94

Chapter 5 NPY Inhibits the Neuronal Branch of the Stress Response.................................... 95

5.1 Introduction ................................................................................................................ 95 5.2 Results ........................................................................................................................ 97

5.2.1 The cold FST led to a sustained activation of the HPA axis ........................... 97 5.2.2 The cold FST led to a sustained activation of the neuronal pathway .............. 97 5.2.3 Co-localization of NPY and TH in the adrenal medulla ................................. 98 5.2.4 The cold FST increased the level of PNMT-immunoreactivity in

chromaffin cells in vitro ................................................................................... 98 5.2.5 The cold FST increased tyrosine hydroxylase-immunoreactivity in adrenal

slices ................................................................................................................. 99 5.2.6 The cold FST-induced increase of TH-ir was reversible in adrenal slices ...... 99 5.2.7 The loss of NPY did not affect the stress-induced increase in plasma

corticosterone ................................................................................................... 100 5.2.8 The loss of NPY altered the neuronal branch of the stress response ............... 100 5.2.9 Expression of NPY Y receptors in the adrenal medulla .................................. 101 5.2.10 Injection of BIBP3226 a NPY Y1 antagonist was associated with an

increase in adrenal TH-ir .................................................................................. 101 5.2.11 Injection of BIIE0246 an NPY Y2 antagonist or L152,804, an NPY Y5

antagonist, did not alter adrenal TH-ir ............................................................. 102 5.2.12 Injection of BIIE0246 was associated with an increase in adrenal NPY-ir... 103 5.2.13 Injection of BIBP3226 or L152,804 did not alter adrenal NPY-ir ................ 103

5.3 Discussion .................................................................................................................. 137 5.3.1 NPY inhibits the expression of adrenal TH and NPY. .................................... 137 5.3.2 NPY does not completely suppress the stress response. ................................. 138 5.3.3 Possible mechanisms underlying the regulatory role of NPY in controlling

the levels of adrenal TH and NPY. ................................................................... 139 5.3.4 Working hypothesis of how adrenal NPY modulates adrenal activity in

the stress response. ........................................................................................... 141 5.4 Conclusion ................................................................................................................. 142

Chapter 6 Discussion .............................................................................................................. 145

6.1 Role of NPY in the cardiovascular system................................................................. 146 6.1.1 Role of peripheral NPY in cardiovascular modulation ................................... 146 6.1.2 Role of the adrenal medulla in cardiac function .............................................. 147 6.1.3 Role of central NPY in the cardiovascular system .......................................... 148

6.2 Role of NPY in metabolic regulation ......................................................................... 148 6.2.1 Role of central NPY in feeding ....................................................................... 148 6.2.2 Role of peripheral NPY in metabolic regulation ............................................. 149

6.3 Role of NPY in the stress response ............................................................................ 150

viii

6.3.1 Role of NPY in controlling the two major stress pathways in the PNS .......... 150 6.3.2 Peripheral NPY mediates stress-induced hypertension ................................... 151 6.3.3 Antagonistic effect of central and peripheral NPY in the cardiac response

may modify the stress response ........................................................................ 152 6.3.4 Interaction between NPY and catecholamines in cardiovascular

modulation may be a survival mechanism in response to stress ...................... 152 6.3.5 Peripheral NPY mediates stress-induced obesity ............................................ 154

6.4 Future directions ........................................................................................................ 155

Appendix A .............................................................................................................................. 156

Appendix B .............................................................................................................................. 157

Appendix C .............................................................................................................................. 158

References ................................................................................................................................ 161

ix

List of Figures

Figure 1-1. Processing pathway of NPY. ................................................................................. 6

Figure 3-1. Schematic of NPY fluorescent fusion protein constructs. ..................................... 36

Figure 3-2. Expression of wild type and mutant prohormones. ............................................... 38

Figure 3-3. Venus fluorescent signals were directly detected on the SDS-PAGE gel using

a Typhoon 8600 gel scanner. ........................................................................................... 40

Figure 3-4. Wild type and L7P NPY prohormones were sorted into the regulated

secretory pathway in AtT-20 cells. .................................................................................. 42

Figure 3-5. L7P and wild type prohormones were packaged into the same dense core

granules in AtT-20 cells. .................................................................................................. 44

Figure 3-6. Mutant and wild type prohormones were differentially co-packaged into

single dense core granules in AtT-20 cells....................................................................... 46

Figure 3-7. Co-expression and co-localization of NPY and FMRFamide in AtT-20 cells. ..... 48

Figure 3-8. NPY T1128 polymorphism leads to an increase in neuropeptide secretion. ......... 50

Figure 4-1. The two major peripheral stress pathways. ........................................................... 57

Figure 4-2. The cold FST activated the HPA axis ................................................................... 67

Figure 4-3. The cold FST activated the neuronal branch of the stress reponse. ...................... 69

Figure 4-4. NPY (GFP)-expressing neurons and neuroendocrine cells in the nervous

system. ............................................................................................................................. 71

Figure 4-5. NPY-ir in chromaffin cells from wild type and NPY knockout mice. .................. 73

Figure 4-6. The cold FST increased the level of NPY-ir in chromaffin cells in vitro. ............ 75

Figure 4-7. Brief exposure to fox urine increased the level of NPY-ir in chromaffin cells

in vitro. ............................................................................................................................. 77

Figure 4-8. The cold FST increased the level of adrenal NPY-ir in situ. ................................ 79

Figure 4-9. The cold FST reversibly increased the level of adrenal NPY in situ. ................... 81

Figure 4-10. The cold FST increase GFP expression in chromaffin cells from NPY (GFP)

mice. ................................................................................................................................. 83

Figure 4-11. The adrenal levels of NPY mRNA increased 3 hours after the cold FST. .......... 85

Figure 4-12. Expression of PP, PYY and NPY mRNA in the adrenal medulla....................... 87

x

Figure 5-1. Catecholamine biosynthetic pathway. ................................................................... 96

Figure 5-2. The cold FST led to a sustained activation of the HPA axis. ................................ 104

Figure 5-3. The cold FST led to a sustained activation of the neuronal axis of the stress

response. ........................................................................................................................... 106

Figure 5-4. Co-localization of NPY and TH in chromaffin cells. ............................................ 108

Figure 5-5. The cold FST increased PNMT-ir in chromaffin cells in vitro. ............................ 110

Figure 5-6. The cold FST increased the level of adrenal TH-ir in situ. ................................... 112

Figure 5-7. The cold FST reversibly increased the level of adrenal TH-ir in situ. .................. 114

Figure 5-8. The cold FST led to a sustained activation of the HPA axis in NPY knockout

mice. ................................................................................................................................. 116

Figure 5-9. The cold FST did not increase the level of adrenal TH-ir in NPY knockout

mice. ................................................................................................................................. 118

Figure 5-10. Expression of NPY Y receptors in the adrenal medulla and whole brain. .......... 120

Figure 5-11. Blocking the NPY Y1 receptor was associated with an increase in the level

of adrenal TH-ir ............................................................................................................... 122

Figure 5-12. Injection per se was sufficiently stressful that it led to an increase in the

level of adrenal TH-ir. ...................................................................................................... 124

Figure 5-13. Blocking the NPY Y1 receptor in NPY knockout mice did not alter the level

of adrenal TH-ir. .............................................................................................................. 126

Figure 5-14. Blocking the NPY Y2 or Y5 receptor did not affect the level of adrenal TH-

ir. ...................................................................................................................................... 128

Figure 5-15. Blocking the NPY Y2 receptor was associated with an increase in the level

of NPY-ir in the adrenal medulla ..................................................................................... 130

Figure 5-16. Injection per se did not alter the level of adrenal NPY-ir ................................... 132

Figure 5-17. Blocking the NPY Y1or Y5 receptor did not affect the level of adrenal NPY-

ir. ...................................................................................................................................... 134

Figure 5-18. Working model. ................................................................................................... 141

xi

List of Tables

Table 1. Primers, annealing temperature and amplifying cycles for NPY Y receptors RT-

PCR.. ..................................................................................................................... 26

xii

Acknowledgements

My Ph.D. thesis cannot be done without the patient guidance, heart-warming

encouragement, inspirational motivation and continuous support from Dr. Matthew Whim, my

supervisor and mentor. I really appreciate Matthew’s careful supervision in my experiments and

writing during my Ph.D. training. I also thank him for his useful advice on my postdoc seeking

and his care in my career plan. I respect his careful attitude and decent accomplishment in science,

which inspires and guides me to continue with my academic career.

I want to show my thanks to Dr. June Liu for her suggestions and support in my Ph.D.

thesis research and her kindly care in my life. My thanks also go to my committee members, Dr.

Douglas Cavener, Dr. Bernhard Luscher, Dr. Melissa Rolls and Dr. Byron Jones for their help

and advice in my research and dissertation writing.

I acknowledge all the previous and current lab members for their suggestions and help in

my research projects.

Finally I would like to thank my Dad Tiejun Wang, Mom Fusheng Bian and Fiancé

Jiehuan huang for their endless love and support to me.

Chapter 1

Introduction

1.1 Neurotransmitters in the nervous system

The nervous system contains two classes of neurotransmitters. The first group contains

classical neurotransmitters and some examples are acetylcholine, the catecholamines, glutamate

and GABA. The other type includes neuropeptide transmitters such as ghrelin, agouti-related

peptide (AgRP) and neuropeptide Y (NPY). Neuropeptides are often located in the same neurons

as classical transmitters (Hokfelt et al., 1980; Hokfelt et al., 1987). Classical neurotransmitters are

usually packaged into small clear-cored vesicles and released from presynaptic varicosities. In

contrast, neuropeptides are peptides consisting of a few up to a hundred amino acids. They are

synthesized in the cell body and stored in dense core secretory granules (DCSGs). DCSGs can

fuse at the soma, presynaptic and postsynaptic sites outside of synaptic active zones (Brigadski et

al., 2005; Hook et al., 2008; Huang and Neher, 1996; Lochner et al., 2006; Sobota et al., 2010;

Whim, 2006).

As neurotransmitters, neuropeptides mediate cell-cell communication in synaptic

transmission and can regulate the excitability of neurons by depolarization or hyperpolarization of

the membrane potential (Hook et al., 2008). As peptide hormones, neuropeptides are required for

endocrine modulation of central and peripheral targets and can alter gene expression, blood flow

and the secretion of other hormones. The same neuropeptides can play important roles both in

neurotransmission and as circulating hormones (Cavadas et al., 2006; Fu et al., 2004; Hook et al.,

2008; Kuo et al., 2007b; Silva et al., 2001; Urban and Randic, 1984). For example as a

neurotransmitter corticotropin-releasing factor (CRF) increases the excitability of hippocampal

2

neurons (Blank et al., 2003). As a hormone CRF triggers ACTH release from the anterior

pituitary leading to the secretion of glucocorticoids into the blood so altering metabolism during

the stress response (Sapolsky et al., 2000).

1.2 Hypocretin: one example of a prototypical neuropeptide

One example of neuropeptide which has been thoroughly studied is hypocretin (Hcrt)

which is also known as orexin. Hcrt 1 and 2 (also called orexin A and B) are two forms of this

neuropeptide which are derived from the same precursor preprohypocretin which is selectively

expressed in the hypothalamus (de Lecea et al., 1998; Gautvik et al., 1996).

Intracerebroventricular (i.c.v) injection of Hcrt during the light cycle when rodents are least

active promoted wakefulness and induced hyperphagia in rats suggesting a role for Hcrt in

controlling sleeping and feeding (Sakurai et al., 1998; Willie et al., 2001). Hcrt knockout mice ate

significantly less compared to wild type controls (Willie et al., 2001). In addition Hcrt-deficient

mice or both Hcrt 1 and Hcrt 2 receptors double knockout mice displayed a narcolepsy phenotype

(Chemelli et al., 1999; Sakurai, 2007). Hcrt evoked a substantial but reversible increase in the

frequency of excitatory postsynaptic currents in rat hypothalamic neurons indicating the

excitatory role of Hcrt in mediating synaptic transmission (de Lecea et al., 1998). These peptides

activated wake-promoting neurons in the hypothalamus and brain stem to maintain a consolidated

awake state (Rolls et al., 2010; Sakurai, 2007).

Disruption of sleep is strongly associated with impaired glucose metabolism and obesity

risk (Spiegel et al., 2009). Since hypothalamic Hcrt neurons play significant roles in regulating

both metabolism and wakefulness, it is possible that they centrally coordinate metabolism as well

as arousal to maintain homeostasis (Rolls et al., 2010). Hcrt neurons send projections to many

brain regions including the hippocampus, amygdale and ventral tegmental area (VTA) as well as

3

to the pituitary and sympathetic neurons in the spinal cord suggesting that Hcrt may participate in

autonomic regulation and the stress response (Sakurai, 2007; van den Pol, 1999; Willie et al.,

2001). Both central and peripheral injections of Hcrt increased plasma levels of corticosterone

(Hagan et al., 1999; Malendowicz et al., 1999). Hcrt 1 and 2 receptors are expressed in the

adrenal medulla thus Hcrt may affect epinephrine release and in this way modulate vascular tone

(Lopez et al., 1999). This idea is consistent with the finding that Hcrt injection increased heart

rate and blood pressure (Lopez et al., 1999; Shirasaka et al., 1999) .

Many neuropeptides, like Hcrt, are involved in regulating numerous physiological

processes centrally as well as peripherally. Understanding the function of the nervous system

involves determining the mechanisms for controlling neuropeptide synthesis and secretion.

1.3 Distribution of neuropeptide Y in the nervous system

Neuropeptide Y (NPY) is a 36 amino acid peptide which is widely expressed throughout

the central as well as peripheral nervous systems in mammals (Lundberg et al., 1982b; Tatemoto,

1982b; Tatemoto et al., 1982). It is present in many brain regions including the hippocampus,

hypothalamus, amygdala and cortex (Adrian et al., 1983; Allen et al., 1983; Gray and Morley,

1986), the peripheral nervous system including sympathetic post-ganglionic neurons and the

adrenal medulla (Lundberg et al., 1986; Lundberg et al., 1984; Lundberg et al., 1983; Renshaw

and Hinson, 2001) as well as peripheral organs such as the pancreas and spleen (Ericsson et al.,

1987; Whim, 2011). The primary structure of human NPY differs from porcine NPY in only one

amino acid (Corder et al., 1984; Tatemoto et al., 1982). Studies have revealed that NPY is

strongly conserved throughout evolution in many species from frogs, birds, rodents to humans

(Hoyle, 1999; Larhammar et al., 1993) and this indicates an important physiological function of

NPY.

4

In the central nervous system, substantial amounts of NPY are found in hypothalamic

nuclei including the arcuate nucleus, paraventricular nucleus and dorsomedial nucleus (Allen et

al., 1983; Chronwall, 1985). In 1983 it was first reported that NPY-like immunoreactivity was

present in catecholamine neurons (adrenergic neurons) in the human medulla oblongata (Hokfelt

et al., 1983). Since then other studies have shown that not only catecholamines but also a variety

of other neurotransmitters such as GABA or neuropeptides such as AgRP coexist with NPY

(Aoki and Pickel, 1990; Morton and Schwartz, 2001). Therefore NPY may function in the brain

by interacting with other modulators.

Peripherally it was first demonstrated in 1982 that NPY-like immunoreactivity was found

within neurons that contained tyrosine hydroxylase (TH, the initial and rate-limiting enzyme for

catecholamine biosynthesis) and dopamine-beta-hydroxylase (DβH, an enzyme downstream of

TH which is also required for catecholamine production) (Kvetnansky et al., 2009; Lundberg et

al., 1982b). NPY has been found in peripheral sympathetic neurons and is co-stored and co-

released with the catecholamines (Bischoff and Michel, 1998; Lundberg et al., 1983; Mormede et

al., 1990; Whim, 2006). NPY is also found in peripheral non-neuronal tissues such as the spleen

(Ericsson et al., 1987). This distribution suggested that there are potential interactions between

NPY and adrenergic systems in modulating peripheral physiology.

1.4 Processing and biosynthesis of neuropeptide Y

NPY is derived from a larger protein precursor, the 69 amino-acid peptide

proneuropeptide Y (proNPY). The precursor hormone is first expressed as a 97 amino-acid

peptide preproneuropeptide Y (preproNPY) and the 28 amino-acid signal sequence of preproNPY

is co-translationally removed by cleavage by a membrane-bound signal peptidase when the

preproNPY translocates into the ER lumen. The proNPY is transported to the cis-Golgi network

5

and then from the trans-Golgi network, proNPY is packaged into DCSGs where processing

enzymes including prohormone convertase 2 (PC-2) or prohormone covertase 1/3 (PC 1/3),

peptidylglycine α-amidating monooxygenase (PAM) and carboxypeptidase E (CPE) are co-

packaged (Marx et al., 1999). PC-2 in the superior cervical ganglion neurons or PC 1/3 in the

AtT-20 cell line cleaves the carboxyl-terminal peptide of NPY (CPON)(Paquet et al., 1996). CPE

removes the basic residues and PAM amidates the peptide intermediate to generate the

biologically active 36 amino acid peptide, NPY (Cerda-Reverter and Larhammar, 2000; Dikeakos

and Reudelhuber, 2007; Hegde and Bernstein, 2006; Hook et al., 2008; Marx et al., 1999)(Figure

1-1). Although CPON has been shown to be co-stored and co-released with NPY, no biological

function has been demonstrated for this peptide (Allen et al., 1987; Balbi and Allen, 1994).

NPY secretion from neurons is evoked by action potentials and is released via the

regulated secretory pathway (in contrast to the constitutive secretory pathway)(Dikeakos and

Reudelhuber, 2007; Hexum et al., 1987; Lou et al., 2005; Mitchell et al., 2008; Ramamoorthy and

Whim, 2008; Rudehill et al., 1992; Whim, 2006; Whim and Lloyd, 1989; Zhang et al., 2010).

6

Figure 1-1. Processing pathway of NPY.

NPY is synthesized as a precursor with the signal sequence in the N terminus. The signal

sequence is cleaved by a signal peptidase co-translationally when the preproNPY translocates into

the ER lumen. ProNPY is packaged into DCSGs via the trans-Golgi network. In DSCGs, CPON

is removed by PC 2 (or PC 1/3 in some cells such as AtT-20 cells). The intermediate is amidated

by PAM and two basic residues at the C terminal end of NPY are removed by CPE to generate

the mature NPY.

7

1.5 Physiological functions of neuropeptide Y

The related Y family members include pancreatic polypeptide (PP) which is produced

mainly by the pancreas and peptide YY which is synthesized by cells in the small intestine and

the pancreas. All Y family members share similar peptide sequence and can activate all NPY

receptors (Larhammar, 1996; Larhammar et al., 1993; Lin et al., 2004; Lundberg et al., 1982a;

Tatemoto, 1982a, b; Tatemoto and Mutt, 1980).

The known NPY receptors include Y1, Y2, Y4, Y5 and in the mouse also y6. All Y

receptors are G protein coupled receptors and their activation usually causes inhibitory responses

such as the inhibition of cAMP accumulation. NPY and PYY have similar affinity for all four

functional Y receptors. However PP preferentially activates Y4 receptors (Dumont et al., 1992;

Lin et al., 2004; Michel et al., 1998; Pons et al., 2008).

NPY is implicated in diverse physiological functions including metabolic regulation,

anxiety, depression, seizure, alcoholism and cardiovascular modulation (Abe et al., 2007; Arora

and Anubhuti, 2006; Erickson et al., 1996; Feder et al., 2009; Kuo et al., 2007b; Leggio et al.,

2011; Lin et al., 2004; Nguyen et al., 2011; Palmiter et al., 1998; Rotzinger et al., 2010; Thorsell,

2010).

1.5.1 NPY in the cardiovascular system

In 1982 the group that first isolated NPY from porcine brain found that injection (i.p.) of

this new peptide induced prolonged vasoconstriction. The effect was resistant to α-adrenoceptor

blockade and was not abolished in sympathectomized animals, indicating a direct action of NPY

on vascular smooth muscle. This was the first report of the biological activity of NPY and

suggested the role of peripheral NPY in regulating the cardiovascular system (Lundberg and

8

Tatemoto, 1982). The next year it was shown that central administration of NPY induced

hypotension in the rat so providing evidence of the involvement of central NPY in also regulating

the cardiovascular system (Fuxe et al., 1983). Since then a growing literature has confirmed that

NPY plays an important role in the cardiovascular system.

Conventional NPY transgenic rats that over-express NPY under the control of

endogenous regulatory elements showed decreased blood pressure and reduced catecholamine

release suggesting the role of endogenous NPY in the long-term inhibition of the cardiovascular

system (Michalkiewicz et al., 2003). This is consistent with the effect of central injection of NPY

leading to hypotension (Fuxe et al., 1983). This central NPY-induced hypotension was primarily

mediated by a presynaptic Y2 receptor inhibition (Chen and Westfall, 1993).

Conditional transgenic mice over-expressing NPY in adrenergic and noradrenergic

neurons (i.e. brain noradrenergic neurons and peripheral sympathetic neurons) displayed stress-

induced hypertension indicating a stimulatory role of peripheral NPY in the cardiac response

(Ruohonen et al., 2008). This is also consistent with the effect of injection (i.p) of NPY in

inducing vasoconstriction (Lundberg and Tatemoto, 1982).

Moreover peripheral NPY causes a prolonged vasoconstriction not only by directly

activating NPY Y1 receptors but also potentiates norepinephrine-evoked vasoconstriction via an

increase in intracellular calcium (Abe et al., 2010; Fallgren et al., 1993). The NPY Y1 receptor-

mediated effect was confirmed when it was found that Y1 receptor knockout mice showed an

absence of the NPY-induced increase of blood pressure (Pedrazzini et al., 1998).

In addition peripheral NPY promotes vascular remodeling including angiogenesis. This is

mediated predominantly by Y2/Y5 receptors as well as by vascular smooth muscle proliferation

and hypertrophy via Y1 receptors (Kuo et al., 2007a; Lin et al., 2004; Pedrazzini et al., 1998;

Pons et al., 2004).

9

Since stress triggers the release of NPY together with norepinephrine from sympathetic

nerves, and NPY receptors as well as adrenergic receptors are expressed in heart, NPY and

norepinephrine are believed to be mediators of stress-induced hypertension (Abe et al., 2010;

Chottova Dvorakova et al., 2008).

1.5.2 NPY in feeding and metabolism

In 1984 NPY was found to promote hyperphagia. The first evidence was that i.c.v

administration of NPY significantly induced feeding behavior in rats (Levine and Morley, 1984;

Stanley and Leibowitz, 1984). Since then more evidence has accumulated showing that NPY is

involved in regulating food intake, energy homeostasis as well as body weight.

In the central nervous system, NPY is most highly co-expressed with AgRP in the

hypothalamic arcuate nucleus (ARC), the neural center involved in feeding control and

integration of the signaling of energy balance (Arora and Anubhuti, 2006; Funahashi et al., 2000;

Kotz et al., 1998; Lin et al., 2006; Nguyen et al., 2011; Pedrazzini et al., 1998; Raposinho et al.,

2001; Stanley et al., 1989; Wilding, 2002). These NPY/AgRP neurons in the ARC are major

targets of the food-stimulating hormone ghrelin as well as the food-inhibiting hormones leptin

and insulin via their distinctive receptors (Arora and Anubhuti, 2006; Wilding, 2002). Ghrelin

increases feeding behavior by stimulating the production of NPY and AgRP in the ARC whereas

leptin and insulin inhibit food intake and body weight by decreasing the expression of NPY and

AgRP (Air et al., 2002; Greenman et al., 2004; Kim et al., 2006; Morrison et al., 2005; Nogueiras

et al., 2008; Schwartz et al., 1992; Zigman and Elmquist, 2003).

Using optogenetic manipulation, a recent study has shown that directly stimulating these

NPY/AgRP neurons is sufficient to induce food intake thus providing direct evidence of the

central role of these NPY neurons in regulating feeding behavior (Aponte et al., 2011).

10

In the peripheral system, NPY is thought to directly act on white fat tissue via Y2

receptor. Stress increased NPY/Y2 signaling and thus caused fat cell growth, which resulted in

obesity (Kuo et al., 2008; Kuo et al., 2007b).

1.5.3 NPY in emotional regulation

In 1987 it was found that stress-induced gastric erosion was reduced by approximately 50%

after i.c.v injection of NPY in rats, suggesting an anti-stress role of NPY (Heilig and Murison,

1987). In 1988 Heilig et al. found that anti-depressant drugs increased NPY immunoreactivity in

the brain, and then in 1989 the same group reported that i.c.v administration of NPY showed an

anxiolytic-like effect by interacting with noradrenergic systems in rat anxiety models (Heilig et

al., 1989; Heilig et al., 1988). NPY is considered to be a potent anti-depressant and anxiolytic,

and the role of NPY in regulating emotionality has been studied (Feder et al., 2009; Heilig, 2004;

Morales-Medina et al., 2010; Redrobe et al., 2002; Rotzinger et al., 2010; Sajdyk et al., 2004).

Central administration of NPY inhibits the development of fear conditioning and

promotes extinction whereas NPY Y1 antagonists act to increase the fear response in rats

(Gutman et al., 2008). Furthermore injection of NPY into the amygdala increased resilience to

stress and decreased anxiety-like behaviors in response to acute stress in rats (Sajdyk et al., 2008).

NPY also counteracts the effect of CRH which is expressed in the hippocampus, hypothalamus

and amygdala and promotes anxiety, so maintaining emotional homeostasis during the stress

response (Sajdyk et al., 2004).

11

1.5.4 NPY in epilepsy

The hippocampus is an epileptogenic brain region. NPY was shown to act presynaptically

on hippocampal CA1 neurons to inhibit glutamate-mediated synaptic transmission onto pyramidal

neurons (Colmers et al., 1987). NPY synthesis and release was markedly up-regulated in

hippocampal neurons during epilepsy in rats first suggesting a role of NPY in modulating limbic

excitability and seizure (Marksteiner et al., 1990; Pitkanen et al., 1989).

Mild seizures occurred in 30% of NPY knockout mice but not in littermate wild type

animals. When challenged with the GABA antagonist, pentylenetetrazole (PTZ), NPY-deficient

mice were hyperexcitable and more susceptible to seizures. These results from NPY knockout

mice indicated the essential role of NPY in reducing the levels of excitability in the central

nervous system and in protection from seizures (Erickson et al., 1996).

NPY Y5 receptor knockout mice were found to be more sensitive to kainic acid-induced

seizures and the inhibitory effects of applied NPY in hippocampal slices were absent from these

knockout mice. These data suggested that Y5 receptors were involved in the inhibitory actions of

NPY in the hippocampus (Marsh et al., 1999).

1.6 My thesis research on neuropeptide Y

NPY is abundantly expressed all over the body and is involved in numerous physiological

processes (Abe et al., 2007; Arora and Anubhuti, 2006; Erickson et al., 1996; Feder et al., 2009;

Kuo et al., 2007b; Leggio et al., 2011; Lin et al., 2004; Nguyen et al., 2011; Palmiter et al., 1998;

Rotzinger et al., 2010; Thorsell, 2010). Therefore determining the mechanisms involved in the

regulation of NPY expression and secretion will help us to understand the functioning of the

nervous system. In contrast to central NPY, less attention has been placed on the role of

12

peripheral NPY although NPY is released from sympathetic neurons as well as the adrenal

medulla, and NPY receptors are expressed in peripheral tissues including the heart, liver,

pancreas and adipose tissue (Kuo et al., 2007b; Moltz and McDonald, 1985; Rimland et al., 1991;

Sheriff et al., 1990; Whim, 2006, 2011). My Ph.D. thesis research has been involved in

investigating the regulation and role of peripheral NPY in the sympathetic nervous system.

A single nucleotide polymorphism (SNP) that changes the seventh amino acid in the

signal sequence of the NPY preprohormone (preproNPY) was identified in 1998 (Karvonen et al.,

1998). Humans carrying this T1128 polymorphism had higher levels of plasma NPY after

exercise and this SNP was associated with higher serum cholesterol levels and increased risk for

metabolic and heart diseases (Heilig et al., 2004; Karvonen et al., 1998; Karvonen et al., 2001;

Nordman et al., 2005; Pesonen, 2006, 2008). The data above suggested a role of the peripheral

NPY in modulating metabolism.

Since the location of the identified SNP T1128C is in the preproNPY signal sequence, it

was suggested to lead to a change in the trafficking, synthesis and/or secretion of NPY (Kallio et

al., 2001; Karvonen et al., 1998). I directly tested this hypothesis by comparing the expression of

NPY prohormones derived from mutant and wild type preproNPY. I found that this SNP did not

prevent synthesis and trafficking of the prohormone, but altered the biosynthesis and secretion of

NPY, which suggested a molecular mechanism that may underlie the T1128C phenotype (Heilig

et al., 2004; Karvonen et al., 1998; Karvonen et al., 2001; Nordman et al., 2005; Pesonen, 2006,

2008).

Stress is known to up-regulate sympathetic activity and the stress-induced increase of

peripheral NPY exaggerates high fat diet induced-obesity (Kuo et al., 2007b). This study by Kuo

et al. directly linked sympathetic NPY with the stress response and metabolic changes. As

modified postganglionic neurons and as part of the sympathetic system, the adrenal medulla can

be activated by stress (Hiremagalur et al., 1994; Nankova et al., 1996; Nankova and Sabban, 1999;

13

Sabban et al., 2006). The adrenal glands consist of a cortex and medulla and are part of the

mechanism that allows the organism to cope with stress (Carrasco and Van de Kar, 2003).

The adrenal medulla, where abundant NPY is expressed, is activated during the stress

response (Colomer et al., 2010; Colomer et al., 2008; Ulrich-Lai and Engeland, 2002; Varndell et

al., 1984). Therefore the other aim of my thesis was to investigate how stress modulated adrenal

NPY and the physiological roles of NPY in regulating adrenal functions. My finding suggests that

acute stress increases the levels of adrenal NPY and NPY plays an important role in inhibiting the

neuronal branch of the stress response which may act to prevent a pathological activation of the

fight-or-flight response.

Chapter 2

Materials and Methods

2.1 Specific Methods for Chapter 3

2.1.1 Site-directed mutagenesis

Site-directed mutagenesis was conducted on a pcDNA3.1 vector (Invitrogen, Carlsbad,

CA) which contained preproNPY/fluorescence tag/ FMRFamide using custom-designed

mutagenic primers with the Quick-Change system (Stratagene, La Jolla, CA). The mutant

constructs that used the most frequently employed proline condon CCC were termed “L7P”. This

mutation was generated by using the forward primer:

5’-GCTAGGTAACAAGCGACCCGGGCTGTCCGGACTG-3’

and the reverse primer:

5’-CAGTCCGGACAGCCCGGTCGCTTGTTACCTAGC-3’.

The mutation sites are underlined.

The mutant constructs which contained the actual polymorphism observed by Karvonen et al.

(1998) are referred to “T1128C”. This mutant changed CTG to CCG, a rarely used codon for

proline and it was made with the forward primer:

5’- GCTAGGTAACAAGCGACCGGGGCTGTCCGGACTG-3’

and reverse primer:

5’- CAGTCCGGACAGCCCGGTCGCTTGTTACCTAGC- 3’.

The mutation site is underlined.

15

The constructs were confirmed by sequencing. The primers and sequencing were performed by

the PSU nucleic acid facility (University Park, PA).

2.1.2 AtT-20 cells culturing

AtT-20 cells were grown in 50 ml flasks in 6 ml DMEM containing 10% FCS

(Mediatech, Herndon, VA) and split 1:6 every three days or 1:10 every five to seven days. For

experiments AtT-20 cells were cultured in 35 mm dishes. One hundred microliters of a 1:1 split

cells was plated onto a glass coverslip which was pre-coated with poly-D-lysine (Sigma-Aldrich,

St. Louis, MO) and the dish was flooded with 2 ml DMEM/10%FCS. The cells were maintained

in a humidified incubator with 95% O2/ 5% CO2 at 37OC.

2.1.3 Transfection of AtT-20 cells

Plasmid concentrations were measured using a NanoDrop spectrophotometer. AtT-20

cells were transfected with 100 ng of the plasmids using Lipofectamine 2000 (Invitrogen,

Carlsbad, CA) 24 hours after plating.

2.1.4 Immunoprecipiation and Western blot

AtT-20 cells were transfected with wild type or mutant Venus tagged NPY constructs and

then lysed 48 hours after transfection with 100 μl whole cell extraction buffer (WCEB: 50 mM

Tris-HCl with pH 8.0, 120 mM NaCl, 0.5% v/v NP-40, 10% v/v glycerol, 0.2 mM EDTA, 2 mM

EGTA, 1 mM PMSF and a protease inhibitor mix with 0.1 μg/ml antipain, 1 mU/ml aprotinin ,

0.1 μg/ml chymostatin, 0.1 μg/ml pepstatin A, 0.1 μg/ml leupeptin,). In some of the experiments,

16

the crude cell lysates were subject to Western blot. In other experiments using

immunoprecipitation, lysates were incubated with a mouse monoclonal GFP antibody (1:100;

Abgent, #AM1009a) for 3 hours at 4 OC, followed by incubation for 2 hours with protein G

sepharose beads (Sigma, St. Louis, MO). The beads were then centrifuged at 13,200 rpm for 2

minutes and the pellets were washed twice with WCEB.

The crude lysates or immunoprecipitates were boiled with SDS loading buffer for 5

minutes at 95OC. The boiled samples were separated by electrophoresis on a 15% SDS-PAGE gel,

transferred onto the PVDF membranes, and then immunoblotted with a rabbit polyclonal anti-

GFP antibody (1:1500; Rockland Immunochemicals, #18560). Proteins were detected by a goat

anti-rabbit Cy5 secondary antibody (1:2500, Amersham ECL Plex, #PA45011). Fluorescent

signals were scanned with a Typhoon 8600 scanner and the intensity of the detected bands was

quantified using Image-Pro Plus 5.1 software (Media Cybernetics). Statistical significance was

calculated with a one way ANOVA.

The details of the solutions are listed in Appendix C.

The SDS-PAGE gel with the boiled crude lysates was also directly scanned with a

Typhoon 8600.

2.1.5 Immunocytochemistry

AtT-20 cells were transfected with fluorescence- tagged wild type or mutant constructs

for 24 hours and then fixed in 4% paraformaldehyde (in PBS) for 20 min. After 15 min of

permeablization in 0.3% triton-100X (in PBS), cells were incubated with blocking solution (0.25%

BSA and 0.05% Triton-X in PBS) for 30 min and then were incubated with a primary antibody

overnight at 4OC. Primary antibodies included rabbit anti-NPY (1:200; Peninsula Laboratories,

T4070), mouse anti-GM130 (1:1000; BD Transduction Laboratories, #610822), mouse anti-

17

carboxypeptidase E (CPE) (1:600; BD Transduction Laboratories, #610758) and rabbit anti-

FMRFamide (1:200; Peninsula Laboratories, #T4322). Following the incubation with a primary

antibody, cells were washed with PBS for 4 times and then stained with a secondary antibody for

90 minutes, which was followed by washing for 4 times and mounting on a glass slide. Secondary

antibodies were donkey anti-rabbit Alexa 488 (1:200; Invitrogen), goat anti-mouse fluorescein

(FITC), donkey anti-mouse tetramethylrhodamine isothiocyanate (TRITC), donkey anti-rabbit

TRITC (all 1:100; Jackson ImmunoResearch Laboratories).

In the experiments comparing prohormone copackaging, AtT-20 cells were transfected

with the construct plasmids for 48 hours and then incubated with 10 μM cycloheximide for 1 hour

before the fixation to limit ER-derived fluorescence (Sobota et al., 2006).

Cells were fixed for 20 min and then mounted with mounting medium (Vector

Laboratories, # H-1000).

The details of the solutions are listed in Appendix C.

2.1.6 Image analysis

Images were taken using a Nikon TE2000U microscope with a 60 X (1.4 NA) oil-

immersion objective and a Retiga 1300 monochrome camera. Images and analysis were

performed blind to genotype. For the copackaging experiments, puncta were picked in each

fluorescence channel (Venus and RFP) using the same area of interest (AOI, radius of 0.066 μm).

A punctum was first identified in the green channel using an AOI and then the same AOI was

transferred to the red channel. Twenty puncta were selected from each cell along a process and

ten cells on each coverslip from a dish with the transfected cells were analyzed. Two individual

experiments were done using L7P constructs and one individual experiment used the T1128C

construct. Image-Pro Plus 5.1 (Media Cybernetics), Excel 2003, OriginPro7 and ClampFit (Axon)

18

were used for data analysis. Statistical significance was calculated with one way ANOVA and

Kolmogorov–Smirnov tests. Each experiment contained four sister cultures of matched wild type

and T1128C or L7P transfected AtT-20 cells.

2.2 General Methods for Chapter 4 and Chapter 5

2.2.1 Animals

C57BL/6J, C57BL/6J-NPY(GFP) (van den Pol et al., 2009) and 129S-Nptm1Rpa/J NPY

knockout mice (Erickson et al., 1996) were purchased from the Jackson Laboratory. Most of the

animals were male littermates, the only female littermates used were for one group of cell

cultures used for NPY immunostaining in the experiment using the cold forced swim test (cold

FST) (section 2.2.2, 2.2.3 and 2.2.4), one group used for measuring NPY-ir 1 week after the cold

FST (section 2.2.2 and 2.2.5), one group from a BIBP3226 injection experiment (section 2.3.2)

and one group from a L152, 804 injection experiment (section 2.3.2). Animals were housed on a

12:12 hour light-dark cycle (3:00am-3:00 pm) with ad libitum access to water and standard

laboratory rodent chow. All the procedures were approved by the Animal Care Facility and

Committees in the Pennsylvania State University and Louisiana State University Health Sciences

Center.

2.2.2 Cold water forced swim test

Postnatal P21-P23 littermate mice were individually housed for 3 to 5 days to minimize

the stressful effect of weaning. Considering the effects of social isolation, both control and

experimental littermates were housed individually on the same day before exposing an

19

experimental animal with a stressor. After individual housing, experimental mice were placed in

the cold (4-5OC) water for 5 to 6 minutes (cold water forced swim test, cold FST) (Saal et al.,

2003) and then warmed under a heat lamp. Following recovery the animals were returned to their

home cages. Animals used to measure plasma corticosterone levels were decapitated 30 minutes

after the cold FST when the induction of plasma corticosterone is expected to reach the maximal

level (Richard et al., 2010). For c-Fos immunoreactivity mice were decapitated 1 hour after the

cold FST and for the stress reversible effect on NPY- or TH-immunoreactivity animals were

decapitated 24 hours or 1 week after the cold FST. For all the other experiments, animals were

decapitated 24 hours after the cold FST.

2.2.3 Chromaffin cell cultures

Adrenal glands were obtained from mice and the fat tissue was removed. The cortex was

removed from the adrenal glands and the adrenal medulla was placed in dissection medium

(DMEM/8 mM MgCl2). The adrenal medulla was washed twice with HBSS, then digested with

0.1% collagenase (Sigma, # C9891) for 15 minutes in 37OC incubator with O2 and CO2, and then

treated with 0.1% trypsin bovine type XII S (Sigma, # T2271) for 30 minutes at 37OC. Digested

adrenal medulla was transferred into trituration medium to be triturated gently. Dissociated cells

were pelleted by centrifuging at 1,000 g for 4 minutes. Pellets were resuspended with

DMEM/10%FBS and 100 μl of cell suspension was placed onto poly-D-lysine pre-coated

coverslips in culture dishes and incubated for 45 minutes in the 37OC incubator with O2 and CO2

Then the cells were flooded with 2 ml DMEM/10%FBS and maintained in the 37OC incubator

with O2 and CO2.

The details of the solutions are listed in Appendix C.

20

2.2.4 Immunocytochemistry

Two hours after flooding, chromaffin cells were washed with PBS, fixed with 4%

paraformaldehyde for 20 minutes and then permeabilized with 0.3% Triton-X (in PBS) for 15

minutes. Following permeabilization, cells were incubated with blocking solution (0.25% BSA

and 0.05% Triton-X in PBS) for 30 minutes and then were incubated with a primary antibody at

4OC overnight. The following day, cells were washed with PBS for 4 times, stained with a

secondary antibody for 90 minutes, washed with PBS again for 4 times and then mounted on

glass slides. Primary antibodies were rabbit anti-NPY (1:40,000; Peninsula labs, T 4070), guinea

pig anti-PNMT (1: 100; Acris, # EUD 7001), sheep anti-TH (1:200; Millipore, # AB 1542) and

rabbit anti-TH (1:200; Millipore, # AB 152). Secondary antibodies included donkey anti-rabbit

Alexa 488 (1:200; invitrogen), donkey anti-guinea pig DyLight 549, donkey anti-sheep DyLight

549 and donkey anti-rabbit DyLight 549 (all 1:100, Jackson ImmunoResearch Laboratories).

Chromaffin cells from NPY (GFP) animals were fixed for 20 minutes after culturing and

then mounted on glass slides.

The details of the solutions are listed in Appendix C.

2.2.5 Cryostat frozen sectioning

Fat surrounding each adrenal gland was removed and the adrenals were immersed in 4%

paraformaldehyde (in PBS) for overnight fixation at room temperature. The fixed adrenal glands

were washed with PBS and then frozen in dry ice-cold beta-methylbutane for 45 seconds. Frozen

adrenals were then embedded in molds with cryomatrix and kept at -80 OC until use.

21

2.2.6 Immunohistochemistry

Thirty micrometers thick cryo-sections were washed with PBS, fixed with 4%

paraformaldehyde for 20 minutes and then permeabilized with 0.3% Triton-X (in PBS) for 15

minutes. Following permeabilization, sections were quenched with 3% H2O2 for 45-60 minutes

and incubated with blocking solution (blocking reagent in TBS, Perkin Elmer, # FP1020) for 30

minutes. Then sections were stained with a primary antibody at 4 OC overnight. The next day,

sections were washed with PBS-T (0.05% Tween-20 in PBS), incubated with a secondary

antibody peroxidase-conjugated donkey anti-rabbit or anti-sheep IgG (1:500; Jackson

ImmunoResearch Laboratories) for 30 minutes, washed with PBS-T, incubated in TSA-FITC

(Perkin Elmer, # NEL741) or TSA-Cy3 (Perkin Elmer, # NEL 744) for 10 minutes, washed with

PBS-T, rinsed with distilled water and then mounted. The primary antibodies were rabbit anti-

NPY (1:10,000; Peninsula labs, # T4070), sheep anti-TH (1:200; Millipore, # AB 1542), rabbit

anti-TH (1:200; Millipore, # AB 152) and rabbit anti-c-Fos Ab-5 (1:1,000; Calbiochem, # PC38).

2.2.7 Image analysis

All slides were blinded by a non-experimenter to avoid any bias in the analysis. Images

were taken using a TE2000U microscope with a 10X objective for sections and with a 60X oil

immersion objective for cell cultures. ImagePro Plus 5.1 (Media Cybernetics) was used to

analyze the levels of NPY- or TH-immunoreactivity and GFP fluorescence. For cell cultures, a

circular area of interest (AOI) was used to outline each cell. A polygon area of interest was used

to outline the whole adrenal medulla and part of the adrenal cortex (which is negative for NPY-ir

and was used as a background correction). The mean fluorescence intensity (independent of the

AOI size) was used for analysis.

22

For counting the number of c-Fos-ir positive cells in each adrenal section, a circular AOI

with a radius of 66 μm was used to outline the region. The AOI was moved along the adrenal

medulla to cover the whole medulla (without any areas of overlap) and then the average count

from all AOIs in each adrenal section was recorded for group analysis.

2.2.8 Statistical analysis

Statistical significance was calculated using a matched one sample Student’s t test

(normalized data) or paired Student’s t test (raw data) when two groups of values were compared

(the average values from each individual experiment were compared). The Kolmogorov–Smirnov

test was used for calculating P values in the cumulative fraction data sets (the values from all the

cells in all the independent experiments were plotted in one cumulative graph). Three or four

groups of values were compared using a one-way analysis of variance (ANOVA) post-hoc

Tukey’s paired comparison (the average values from each individual experiment were compared).

2.2.9 Corticosterone ELISA

Five hundred microliters of blood was collected from a decapitated animal and

centrifuged at 1,000g at 4 OC for 15 minutes. Plasma was collected in 1mM EDTA containing

tubes and kept at -80 OC until use. Plasma corticosterone levels were measured using a

commercial EIA kit following the manufacturer’s instructions (Assay Designs, Plymouth Meeting,

PA, #900097). Reactions were read using an absorbance microplate reader at 405 nm with a

correction at 570 nm. A standard curve was generated using standard samples provided by the kit

and validated with a linear regression fit in OriginPro7. Duplicates were measured to calculate the

mean of the optical density (OD) values for each blood sample.

23

2.3 Specific Methods for chapter 4

2.3.1 Fox urine exposure

P19-21 mice were kept in new cages for 3 minutes, exposed to fox urine odor for 5

minutes and then were returned to the home cage (Liu et al., 2010). The animals exposed to fox

urine odor were decapitated 16-24 hours later.

2.3.2 Semi-quantitative RT-PCR of NPY

The adrenal cortex was removed from the whole adrenal gland and the adrenal medulla

was collected. Total RNA was extracted from the adrenal medulla using Trizol (Invitrogen, #

15596-026) and purified with an RNeasy mini Kit (Qiagen, # 74104) following the

manufacturer’s instructions. Sequential dilutions of cDNA’s at 1:4, 1:16 and 1:64 were used for

PCR amplification. NPY expression was normalized by actin expression. The forward NPY

primer was 5’-CAC GAT GCT AGG TAA CAA G-3’ and the reverse primer was 5’-CAC ATG

GAA GGG TCT TCA AG-3’. The forward actin primer was 5’-GCC AAC CGT GAA AAG

ATG AC-3’ and the reverse primer was 5’-CAA CGT CAC ACT TCA TGA TG-3’. The PCR

was run for 4 minutes at 94OC, followed by 45 seconds denaturation at 94

OC, 45 seconds

annealing at 55OC and 1 minute of extension at 72

OC for 35 cycles and an additional cycle of

extension at 72OC for 10 minutes.

2.3.3 RT-PCR of PP and PYY

Total RNA was extracted as describe above (section 2.3.2). The cDNA was obtained by

reverse transcription of 3 ng mRNA from pancreatic islets and of 7 ng mRNA from adrenal

24

medulla. The forward insulin primer was 5’-TTT GTC AAG CAG CAC CTT TG-3’ and the

reverse primer was 5’-GCT GGT AGA GGG AGC AGA TG-3’. The forward NPY primer was

5’-CAC GAT GCT AGG TAA CAA G-3’ and the reverse primer was 5’-CAC ATG GAA GGG

TCT TCA AG-3’. The forward PP primer was 5’-CAC GAT GCT AGG TAA CAA G-3’ and the

reverse primer was 5’-CAC ATG GAA GGG TCT TCA AG-3’. The forward PYY primer was

5’-CAC GAT GCT AGG TAA CAA G-3’ and the reverse primer was 5’-CAC ATG GAA GGG

TCT TCA AG-3’. The PCR was run for 4 minutes at 94OC, followed by 45 seconds denaturation

at 94OC, 45 seconds annealing at 55

OC and 1 minute of extension at 72

OC for 35 cycles and an

additional cycle of extension at 72OC for 10 minutes.

2.4 Specific Methods for chapter 5

2.4.1 Catecholamine ELISA

Five hundred microliters of blood was collected from a decapitated animal and

centrifuged at 1,000g at 4 OC for 15 minutes. Plasma was collected in 1mM EDTA-containing

tubes and kept at -80 OC until use. Plasma epinephrine and norepinerphrine from the same blood

samples were measured using commercial ELISA kits following the manufacturer’s instructions

(RM Diagnostics, # BA E5400). Reactions were read by an absorbance microplate reader at 450

nm with a correction at 620 nm. A standard curve was generated using standard samples provided

by the kit and validated with a linear regression fit in OriginPro7. Duplicates were measured to

calculate the mean of the OD values for each blood sample.

25

2.4.2 Antagonist injection

Littermate animals were intraperitoneal (i.p.) injected with 200 μl saline or BIBP 3226

(Tocris Biosciences, 1mg/kg) 15 minutes before the cold FST. A second group of animals were

i.p injected with 200 μl saline or BIIE 0246 (Tocris Biosciences, 1mg/kg) 15 minutes before the

cold FST. The third group were injected with 200 μl saline or L152, 804 (Tocris Biosciences,

10mg/kg) 15 minutes before the cold FST.

2.4.3 RT-PCR

The hippocampus, hypothalamus, cortex and cerebellum were collected from the whole

brain. Collection of mRNA from the adrenal medulla or brain was as described previously

(section 2.3.2). cDNA obtained by reverse transcription of 20 ng mRNA from the adrenal

medulla or brain was used to amplify NPY Y1, Y2, Y5 and Y6 receptors and cDNA from 60 ng

mRNA was used for the NPY Y4 receptor (Ramamoorthy and Whim, 2008). Primers are

presented in Table1. The PCR was run for 4 minutes at 94OC, followed by 45 seconds

denaturation at 94OC, 45 seconds annealing at the temperatures listed in Table1 and 1 minute of

extension at 72OC for the number of cycles as presented in the Table1 and an additional cycle of

extension at 72OC for 10 minutes.

26

Gene Primers Anealing Tm and amplifying cycles

NPY Y1 Forward: 5’ CGG CGT TCA AGG ACA AGT AT 3’ Reverse: 5’ TGA TTC GCT TGG TCT CAC TG 3’

55OC for 35 cycles

NPY Y2 Forward: 5’ TGC CAA TCT GGT TAG GGA AG 3’ Reverse: 5’ GGT GCC AAC TCC TTG TTC TG 3’

55OC for 35 cycles

NPY Y4 Forward: 5’ TTG CAG TTC TCT GGC TAC CCC TG 3’ Reverse: 5’ CTT GCT ACC CAT CCT CAT CGA 3’

56OC for 35 cycles

NPY Y5 Forward: 5’ CAG ATT AAT CCA GCT GTT CTG C 3’ Reverse: 5’ GAA AAC AGC CTT TAT TTG ACA ATG 3’

55OC for 35 cycles

NPY y6 Forward: 5’ TCA CTA AAT AAG ACC ATC GGG TAG 3’ Reverse: 5’ GGG AGG TTT ACC CTA GGA AAT G 3’

55OC for 10 cycles +

53OC for 30 cycles

NPY Forward: 5’ GCT AGG TAA CAA GCG AAT GGG G 3’ Reverse: 5’ CAC ATG GAA GGG TCT TCA AGC 3’

55OC for 35 cycles

Table 1. Primers, annealing temperatures and amplifying cycles for RT-PCR of NPY Y

receptors.

Chapter 3

A Single Nucleotide Polymorphism Alters the Synthesis and Secretion of

Neuropeptide Y

3.1 Introduction

More than one million single nucleotide polymorphisms (SNPs) are reported to be

distributed throughout the human genome and of these it is estimated that 60,000 are found in

exons. Studying these SNPs is thought to be important because genetic differences between

individuals can have a major impact on the response to disease and environmental factors

(Sachidanandam et al., 2001).

Genome-wide mapping analysis is being used to identify SNPs that are associated with

many kinds of complex diseases such as diabetes, cardiovascular disease, breast cancer, prostate

cancer and bladder cancer (Garcia-Closas et al., 2007; Hunter et al., 2007; Larson et al., 2007;

Saxena et al., 2007; Yeager et al., 2007). Establishing an association between SNPs and disease

susceptibility can help to identify biomedically important genes for diagnosis. This is more

advantageous than conventional gene-hunting and by developing gene-targeted medicine,

personalized therapies will ultimately be possible (Sachidanandam et al., 2001).

Following the identification of a functional SNP, it is important to determine the

molecular mechanisms underlying the correlation between the SNP and the disease it causes. In

the nervous system, gene variants encoding transporters, ion channels and neuromodulators have

been associated with clinical deficits or disorders (Egan et al., 2003a; Karaplis et al., 1995;

Mazei-Robison et al., 2005; Vitko et al., 2005).

28

Among these SNPs, one success story is the identification of the molecular mechanisms

that underlie the brain-derived neurotrophic factor (BDNF) Val66Met polymorphism and its

associated phenotypes. In humans this BDNF polymorphism in the 5’ proBDNF domain is

associated with biopolar disorder, depression, memory impairment and schizophrenia (Egan et al.,

2003a; Egan et al., 2003b; Sen et al., 2003). This functional SNP (valine to methionine) has been

reported to lead to abnormal distribution of the prohormone in both the dendrites and cell body of

hippocampal neurons, and to cause decreased activity-dependent secretion (Egan et al., 2003a).

The abnormal trafficking phenotype seems to be due to impaired binding of the mutant BDNF

with a “sorting” protein sortilin, which has been shown to interact with the prodomain of BDNF

(Chen et al., 2005). Chen et al. have generated a transgenic mouse that endogenously expresses

mutant BDNF. The genetic variant of BDNF containing the SNP alters anxiety-related behaviors

in these mice, which correlates well with the phenotype in humans (Chen et al., 2006).

Another good example is the isolation of a SNP in the signal sequence of the human

preproparathyroid hormone (preproPTH) which was obtained from a patient with familial isolated

hypoparathyroidism. A cysteine residue is substituted with arginine in the hydrophobic core of

the signal sequence of preproPTH, which likely impairs the processing of the preprohormone to

prohormone (Arnold et al., 1990). The impaired processing of the mutant preprohormone is likely

due to inefficient membrane targeting and translocation into the ER, since in vitro experiments

show that the mutation disrupts the interaction of the preprohormone with the signal recognition

particle and the translocon. These in vitro results suggest a molecular mechanism that could

explain the pathophysiologic consequences of this SNP in the patient. This disease is

characterized by hypocalcemia which is due to a lack of functional circulating parathyroid

hormone (PTH), the major calcium-regulating hormone (Karaplis et al., 1995).

A functional SNP which changes the seventh amino acid from leucine (Leu) to proline

(Pro) in the signal sequence of the NPY preprohormone was identified by Karvonen et al. in 1998

29

(Karvonen et al., 1998). This SNP is associated with an increased serum cholesterol level, type 2

diabetes as well as heart disease (Heilig et al., 2004; Karvonen et al., 1998; Karvonen et al., 2001;

Nordman et al., 2005; Pesonen, 2006, 2008). An elevated level of plasma NPY was found in

humans carrying this T1128C mutation after exercise and a higher amount of the mature NPY

hormone was present in human endothelial cells that contained the mutant genotype possibly

indicating an increase in the efficiency of cleavage of the mutant preproNPY (Kallio et al., 2001).

In addition rats that received an i.c.v injection of the purified mutant signal peptide and mature

NPY had markedly elevated food intake compared with those animals injected with the wild type

signal peptide and mature NPY suggesting that the signal peptide alone may have biological

activity (Ding et al., 2005). Although these studies provide some evidence showing a differential

effect between the wild type and mutant preproNPY, the underlying molecular and cellular

mechanisms are still not clear.

Since the single nucleotide mutation T1128C is in the signal sequence of the NPY

preprohormone, I hypothesized that this SNP does not change the sequence of the mature NPY,

but may alter the synthesis, sorting and/or secretion of NPY. To test these possibilities,

fluorescent protein-tagged NPY constructs were expressed in AtT-20 cells, a pituitary cell line. I

then directly tested the above hypothesis by comparing the expression of proNPY derived from

the wild type and mutant preproNPY. My results suggest that proNPY from the wild type and

mutant preproNPY entered the regulated secretory pathway in a similar manner. However

individual dense core granules contained significantly more prohormone derived from mutant

than wild type preproNPY. This polymorphism also increased the secretion of NPY which is

likely due to the increased synthesis. My findings suggest a molecular mechanism that may

underlie the phenotypes associated with the T1128 polymorphism.

3.2 Results

3.2.1 Generation of the wild type and mutant NPY fusion protein constructs

To express and monitor the distribution of NPY prohormone derived from the wild type

and mutant preproNPY, I first generated wild type and mutant fluorescent protein-tagged NPY

constructs (Figure 3-1). The identified T1128C polymorphism changes the DNA sequence from

CTG to CCG (Karvonen et al., 1998). I termed this mutant “T1128C”. The wild type NPY

preprohormone has CTG which is a frequently used leucine codon whereas the T1128C mutant

contains CCG which is the least frequently used proline codon. To minimize any potential effects

caused by the difference in codon usage and examine the solely effect of the amino acid

substitution, I also made additional mutant constructs using the most frequently proline codon

(CCC) and this mutant was termed “L7P”. I used these constructs to examine their expression and

monitor their intracellular trafficking pathways in the following experiments.

AtT-20 cells are an endocrine cell line derived from a mouse pituitary tumor. They do not

endogenously express NPY but are capable of synthesizing NPY (Dickerson et al., 1987). The

advantage of using AtT-20 cells is that they contain dense core granules that can be identified

clearly (Quinn et al., 1991; Sobota et al., 2006).

3.2.2 Expression of NPY prohormones derived from wild type and mutant preproNPY

To determine whether the mutation affected the processing or expression levels of the

NPY prohormone, I used Western blotting to test the expression of proNPY derived from wild

type and mutant preproNPY in AtT-20 cells. The cells were transfected with the wild type NPY-

31

Venus, L7P NPY-Venus or T1128C NPY-Venus. The cell lysates were immunoblotted with a

GFP antibody and the two bands shown correspond to proNPY (37kD) and mature NPY (31kD)

in both wild type and mutant NPY-Venus transfected cells (Figure 3-2 A). The presence of Venus

in each cleavage product was also confirmed by directly scanning a gel for the presence of a

fluorescent protein (Figure 3-3). Therefore the polymorphism did not prevent the synthesis of the

NPY prohormone and mature peptide.

The ratio of NPY vs. proNPY was quantified and no detectable difference was found

between hormones from wild type and mutant preproNPY (Figure 3-2 B). When the tagged

peptides were purified by immunoprecipition and then detected by immunoblotting with a GFP

antibody, only one band was found which corresponded to proNPY (Figure 3-2 C upper band).

The intensity of the bands corresponding to proNPY was normalized by an unspecific band

(Figure 3-2 C lower band). Quantitative analysis showed no detectable difference among all the

groups (Figure 3-2 D). Therefore the polymorphism did not change the expression levels of

proNPY and the relative ratios between mature NPY and the prohormone.

3.2.3 NPY prohormones derived from L7P and wild type preproNPY were sorted similarly

in AtT-20 cells

AtT-20 cells were transfected with the wild type or L7P IRES-GFP constructs and the

transfected cells were identified by cytoplasmic GFP expression. The cells expressing

prohormones derived from wild type or mutant preproNPY showed punctuate NPY-

immunoreactivity (NPY-ir) when stained with an NPY antibody (Figure 3-4 A). When the cells

were transfected with wild type NPY-RFP or L7P NPY-RFP, they showed punctuate distribution

of RFP that co-localized with the NPY-ir (Figure 3-4 B) indicating that these fluorescent fusion

proteins were reliable markers for tracking the distribution of the prohormones.

32

Neuropeptides like NPY usually enter the regulated secretory pathway via the

endoplasmic reticulum (ER), trans-Golgi network and dense core granules (Dikeakos and

Reudelhuber, 2007; Hexum et al., 1987; Rudehill et al., 1992). To test whether the SNP affects

the trafficking pathway of the NPY prohormone, AtT-20 cells were transfected with the wild type

NPY-Venus or L7P NPY-Venus and immunostained with a GM130 (cis-Golgi marker) antibody.

Both wild type NPY-Venus and L7P NPY-Venus co-localized with the GM130-ir (Figure 3-4 C).

Furthermore when the cells were transfected with the wild type NPY-RFP or L7P NPY-RFP,

their punctuate distribution overlapped precisely with carboxypeptidase E-ir (CPE-ir; Figure 3-4

D). CPE is a dense core granule marker and is required for the cleavage of many peptide

prohormones (Fricker, 1988; Hook et al., 2008). Thus prohormones derived from L7P and wild

type preproNPY were targeted into the regulatory secretory pathway.

3.2.4 NPY prohormones derived from L7P and wild type preproNPY entered the same

dense core granules

Most of the NPY SNP carriers are heterozygous thus NPY neurons in vivo are likely to

contain both wild type and mutant preprohormones (Karvonen et al., 1998). To mimic this

situation AtT-20 cells were co-transfected with wild type NPY-Venus and L7P NPY-RFP. Co-

transfection of the cells with wild type NPY-Venus and wild type NPY-RFP was used as a

control. A complete overlap of the Venus and RFP puncta was found in both cases indicating that

prohormones derived from wild type and L7P preproNPY are co-packaged into the same dense

core granules. A two channel intensity line scan confirmed the overlap (Figure 3-5).

From these results, the processing of prohormones from wild type and L7P preproNPY

were indistinguishable. This is in contrast to the effects of SNPs in some other peptides such as

BDNF (Egan et al., 2003a; Karaplis et al., 1995). However sometimes differences in peptide

33

packaging may be quantitative. This possibility was examined in the following series of

experiments.

3.2.5 Prohormones derived from L7P and wild type preproNPY were differentially co-

packaged

AtT-20 cells were co-transfected with wild type and mutant preprohormones tagged with

either Venus or RFP (four possible combinations). Because Venus and RFP might be synthesized

at different intrinsic rates, the control cells were co-transfected with wild type NPY-Venus and

wild type NPY-RFP or with L7P NPY-Venus and L7P NPY-RFP. I measured the intensity of

single puncta in both the green and red channels, and then plotted the data as a cumulative

distribution of the green vs. red ratio (Figure 3-6 A). As shown in the cumulative fraction figure,

the two controls (i.e. wild type NPY-Venus + wild type NPY-RFP vs. L7P NPY-Venus + L7P

NPY-RFP) overlapped, which validated the approach and confirmed that swapping the

prohormones did not alter the synthesis rate of Venus and RFP. The most right-shifted curve with

the “greenest” puncta was from the cells co-transfected with L7P NPY-Venus and wild type

NPY-RFP. Therefore the NPY prohormone from L7P preproNPY accumulated to a higher level

than the NPY prohormone from wild type preproNPY in single granules. On the other hand, the

most left-shifted curve with the “reddest” puncta was from the cells expressing L7P NPY-RFP

and wild type NPY-Venus so these cells contained relatively more L7P-RFP. Thus, I can

conclude that the dense core granules contain higher levels of NPY prohormone derived from

L7P preproNPY than that from wild type preprohormone.

As mentioned in section 3.2.1 and Figure 3-1, the actual observed polymorphism in NPY

is “T1128C” (i.e. CTG mutated to CCG) rather than “L7P” ( i.e. CTG mutated to CCC). I used

the L7P constructs in most of my studies to minimize any potential confounding effects due to

34

codon usage. However in order to test whether the low frequency proline codon (CCG) actually

found in the SNP (i.e. T1128C) had a similar phenotype to L7P, I repeated the experiment with

T1128C-Venus and T1128C-RFP. A similar type of prohormone differential co-packaging was

found as seen in the cumulative distribution (Figure 3-6 B). Therefore the dense core granules

contained higher levels of NPY prohormone derived from mutant preproNPY and this is not due

to codon bias but to the change in the amino acid sequence.

3.2.6 NPY prohormones derived from L7P and wild type preproNPY were sorted similarly

in hippocampal neurons

To examine prohormone trafficking in an acutely isolated cell type, polarized

hippocampal neurons that endogenously synthesize NPY were transfected with both wild type

and mutant preprohormones (Higuchi et al., 1988a; Higuchi et al., 1988b). Similar to the situation

in AtT-20 cells, the SNP did not alter the distribution of the NPY-containing granules but

increased the packaging of NPY prohormone derived from mutant preproNPY in the dense core

granules in hippocampal neurons. The work in this section was mainly contributed by Dr. Prabhu

Ramamoorthy and Mr. Gregory Mitchell. I made the wild type and mutant constructs used for the

neuronal transfections.

3.2.7 Expression of NPY prohormone derived from mutant preproNPY leads to increased

NPY secretion

In order to investigate whether the polymorphism can affect the secretion of NPY,

peptide release was measured from chromaffin cells using the FMRFamide tagging technique.

Chromaffin cells are good model for measuring NPY secretion since these cells endogenously

express and secrete NPY. Chromaffin cells fire action potentials and secretion can be triggered by

35

depolarizing the cells (Whim, 2006; Whim and Moss, 2001). Two copies of FMRFamide were

added to the NPY IRES constructs (Figure 3-1). The release of FMRFamide will activate the

ionotropic FMRFamide receptors that are expressed on the surface of the same cell causing an

inward sodium current. Because the FMRFamide peptide and NPY are co-packaged into the same

dense core granules, the release of FMRFamide is a surrogate for the release of NPY (Whim and

Moss, 2001).

Punctate wild type NPY-Venus or T1128C-Venus was co-localized with FMRFamide-ir

in transfected AtT-20 cells and the overlap was confirmed with an intensity line scan (Figure 3-7).

Thus the FMRFamide peptide and proNPY derived from wild type or mutant preproNPY are

packaged in the same dense core granule and the secretion of FMRFamide can be used to monitor

the release of NPY derived from both wild type and mutant preprohormones.

Chromaffin cells transfected with wild type and mutant NPY-Venus showed punctate

fluorescence confirming the synthesis of proNPY from both wild type and mutant

preprohormones. Chromaffin cells were then transfected with either the wild type or T1128C

NPY preprohormone (IRES constructs, Figure 3-1 and Figure 3-8 A). Since NPY is secreted

through the regulated pathway, stimulation with a train of depolarizations was used to evoke

secretion. The average amplitude of the peptidergic secretory events was larger from the cells

expressing the T1128C NPY preprohormone compared to the wild type NPY preprohormone (72

± 32 pA vs. 29 ± 9 pA, mean ± SD; p < 0.05; Figure 3-8 A and B). The cumulative amplitude

distribution of the release events from the cells expressing T1128C was significantly right-shifted

compared to the NPY peptide derived from wild type preproNPY (Fig 3-8 C and D). These

experiments indicated that the SNP led to an increase in peptide secretion. Transfection of the

chromaffin cells with Venus tagged NPY was done by Mr. Gregory Mitchell; the

electrophysiology was performed by Dr. Matthew Whim; I made the plasmid constructs and

examined the expression of the FMRFamide peptide in NPY-Venus transfected AtT-20 cells.

36

Figure 3-1. Schematic of NPY fluorescent fusion protein constructs.

The SNP is located in the signal sequence (arrows indicate the position of the mutation.)

“T1128C” constructs are the observed polymorphism that changes the 7th amino acid from

leucine to proline (Karvonen et al., 1998). “L7P” constructs contain the same amino acid

substitution but use the most frequently used proline codon. Venus is a yellow fluorescent protein

and RFP is a red fluorescent protein. IRES-GFP expresses cytoplasmic GFP. CPON stands for

carboxyl peptide of NPY. Two copies of the FMRFamide peptide are attached to the end of

CPON. Thanks to the initial L7P constructs generated by Mr. Gregory Mitchell.

37

38

Figure 3-2. Expression of wild type and mutant prohormones.

(A). AtT-20 cells were transfected with NPY preprohormone constructs tagged with Venus. The

bands corresponding to proNPY-Venus (37 kDa) and the cleavage product, NPY-Venus (31 kDa)

are indicated. (B). Processing of NPY prohormone was quantified as the ratio of the cleavage

product to the prohormone. The group data indicates that there is no detectable difference in the

processing ratios (mean ± SD, n = 6, P = 0.19, one way ANOVA). (C). AtT-20 cells were

transfected with wild type NPY-Venus, L7P-Venus, T1128C NPY-Venus or were non-transfected.

The cell lysates were subjected to immunoprecipitation with a GFP antibody. Only the

prohormone (37 kDa) could be detected. (D). NPY synthesis was quantified as the ratio of the

NPY prohormone to the lower non-specific band which was used as a loading control. The group

data indicates that there was no detectable difference in the synthesis of proNPY among the

different constructs (mean ± SD, P = 0.97, one-way ANOVA).

39

40

Figure 3-3. Venus fluorescent signals were directly detected on the SDS-PAGE gel using a

Typhoon 8600 gel scanner.

At the emission wavelength of 520 nm, three fluorescent bands corresponding to preproNPY-

Venus, proNPY-Venus and NPY-Venus can be detected on the gel from the wild type and mutant

NPY-Venus transfected cells but not from the non-transfected cells.

41

42

Figure 3-4. Wild type and L7P NPY prohormones were sorted into the regulated secretory

pathway in AtT-20 cells.

(A). AtT-20 cells were transfected with wild type or L7P-IRES-GFP. Transfected cells identified

by cytoplasmic GFP showed punctuate NPY-ir indicating the synthesis of NPY prohormones

derived from wild type or L7P preproNPY. The lower images showed GFP negative controls.

Scale bar 10 µm. (B). The cells expressing wild type NPY-RFP or L7P NPY-RFP proteins

showed punctuate fluorescence that co-localized with NPY-ir. (C). Wild type NPY-Venus and

L7P NPY-Venus fluorescent fusion proteins partially overlapped with the GM130-ir. (D). Wild

type NPY-RFP and L7P NPY-RFP fluorescence co-localized with CPE-ir. Scale bars 20 µm.

43

44

Figure 3-5. L7P and wild type prohormones were packaged into the same dense core

granules in AtT-20 cells.

Cells were co-transfected with wild type NPY-Venus and wild type NPY-RFP (upper panel) or

with wild type NPY-Venus and L7P NPY-RFP (lower panel). The line scan (right panel) was

through the center of the white boxed region. Scale bar 15 µm.

45

46

Figure 3-6. Mutant and wild type prohormones were differentially co-packaged into single

dense core granules in AtT-20 cells.

(A). The cells were co-transfected with wild type and L7P NPY preprohormones tagged with all

four combinations of Venus and RFP fluorescent proteins. The green vs. red intensity ratio in the

single granules from the co-transfected cells was measured and presented as a cumulative graph.

Twenty puncta were selected from each cell along one process and ten cells were analyzed from

each transfected combination. The graph shown was from a representative experiment (n=2). (B).

Cells were co-transfected with wild type and T1128C NPY preprohormones tagged with all four

combinations of Venus and RFP fluorescent proteins. NS, not significant, **p < 0.01, *p < 0.05,

Kolmogorov-Smirnov test.

47

48

Figure 3-7. Co-expression and co-localization of NPY and FMRFamide in AtT-20 cells.

AtT-20 cells were transfected with wild type NPY-Venus or T1128C NPY-Venus preprohormone.

Both wild type NPY-Venus and T1128C NPY-Venus co-localized with FMRFamide

immunoreactivity (Fa-ir). The line scans (right panels) were taken through the center of the boxed

regions. Scale bar 20µm.

49

50

Figure 3-8. NPY T1128C polymorphism leads to an increase in neuropeptide secretion.

(A). Peptide release was detected using FMRFamide tagging. The transfected chromaffin cells

were identified by cytoplasmic GFP expression. The middle and right panels are examples of the

peptidergic secretory events. Secretion was evoked with a brief depolarization from 80 to 0mV

for 20 ms repeated 200 X at 5 Hz. (B). The average secretory current from the cells expressing

wild type or T1128C-NPY preprohormone (mean ± SD, *p < 0.05, single factor ANOVA). (C).

Cumulative distribution of the peptidergic secretory events from the cells expressing wild type

and T1128C-NPY preprohormones ( p < 0.01, Kolmogorov-Smirnov test). (D). Amplitude

frequency histogram of the secretory events shown in (D).

51

52

3.3 Discussion

In transfected AtT-20 endocrine cells, NPY prohormones derived from wild type and

mutant preproNPY entered the regulated secretory pathway in a similar manner and were co-

packaged in the same dense core granules. However when the levels of proNPY were quantified,

the individual granules were found to contain more NPY prohormone derived from mutant

preproNPY. This is consistent with the observation that the cells expressing the mutant

preprohormone had an increased peptide secretion. TheT1128C polymorphism is therefore a

gain-of-function mutation, which contrasts with many other prohormones such as BDNF which

generally leads to misprocessing and a reduction in secretion (Egan et al., 2003a; Karaplis et al.,

1995).

3.3.1 The SNP might affect the ER trafficking efficiency of the NPY preprohormone

How can the altered biosynthesis and secretion of the hormone by a single amino acid

substitution be explained? The T1128 SNP changes the seventh amino acid of the preproNPY

signal sequence from leucine to proline. However the signal sequence is removed co-

translationally when preproNPY translocates into the endoplasmic reticulum (ER) lumen. In

silico analysis using SignalP suggests that the T1128 polymorphism would not prevent signal

sequence recognition and would not change the cleavage site (Bendtsen et al., 2004). This is

consistent with our finding of the regulated secretion of NPY from the mutant-transfected cells.

Eukaryotic signal sequences typically contain a basic N domain, a hydrophobic core, and

a slightly polar C terminus (Hegde and Bernstein, 2006). Leucine is a hydrophobic amino acid,

however proline falls on the hydrophobic-polar border and the distinctive cyclic structure of the

proline side chain gives proline an exceptional conformational rigidity. A SNP in the signal

53

sequence of preproPTH alters the interaction of the nascent protein with the signal recognition

particle (SRP, ensuring the delivery of the peptide to the ER) and the signal peptidase, which

removes the signal sequence, allowing the preprohormone to translocate into the ER lumen. Like

the mutation in preproPTH, the difference between the two amino acids in the signal sequence of

preproNPY might affect its interaction with the SRP and the signal peptidase (Hegde and

Bernstein, 2006; Karaplis et al., 1995).

The differential packaging of NPY prohormones derived from the wild type and mutant

preproNPY into dense core granules was only detected when the two preprohormones were co-

expressed. This observation may indicate that competition between wild type and mutant

preprohormones during the translocation process might occur. A differential preference for

interaction with the SRP or signal peptidase between the wild type and mutant preproNPY might

account for the different degrees of co-packaging. Signal sequences are multifunctional and can

regulate not only the location of the mature protein but also the efficiency of ER translocation

(Hegde and Bernstein, 2006).

3.3.2 The SNP might cause a more efficient sorting of the NPY prohormone

The postulated bias in the efficiency of ER translocation may lead to a situation in which

the transport of proNPY from mutant preproNPY is faster than wild type within the trans-Golgi

network. This may allow the proNPY from the mutant preprohormone to be sorted into DCSGs

earlier and more efficiently than the wild type.

In addition the signal peptide might have an independent function after cleavage (Hegde

and Bernstein, 2006). It is possible that the mutated signal peptide could facilitate the trafficking

of proNPY.

54

3.3.3 Codon usage does not explain the effect of the SNP

The leucine codon in the wild type preproNPY is CTG which is the most commonly used

version, whereas the T1128C polymorphism, which encodes proline (CCG) is the least frequently

used proline codon. Non-optimal codons in some signal peptides can lead to translational pausing

which helps to correct protein folding and thus increases peptide synthesis (Zalucki et al., 2008). I

found a similar phenotype when I expressed either the optimal codon CCC (L7P) or the non-

optimal proline codon CCG (T1128C) in preproNPY. This suggests that codon usage does not

account for the differential packaging and secretion observed in the NPY prohormone derived

from mutant preproNPY.

3.3.4 Comparison of NPY T1128 with SNPs found in other peptide preprohormones and

prohormones

SNPs have been identified in a variety of peptide prohormones and these are generally

associated with a loss-of-function phenotype. The Val66Met in proBDNF leads to impaired

trafficking and reduced secretion (Egan et al., 2003). The Cys18Arg mutation in the signal

sequence of preproPTH isolated from a patient with hypocalcemia causes inefficient prohormone

processing (Karaplis et al., 1995). The Leu16Arg substitution in the signal sequence of hypocretin

was identified in a human case of early onset narcolepsy and this SNP causes incomplete

prohormone cleavage and retention in the soma (Peyron et al., 2000).

In contrast the T1128C polymorphism led to an increase in the biosynthesis of proNPY

and up-regulation of NPY regulated secretion. These results parallel the finding that humans

carrying this SNP have higher levels of plasma NPY during exercise (Kallio et al., 2001). NPY is

involved in regulating lipid metabolism; for example it can inhibit lipolysis (Turtzo et al., 2001).

Elevation in NPY signaling positively regulates the stress-induced adipocyte growth that

55

develops during obesity (Kuo et al., 2007b). This may contribute to the phenotype of an elevated

serum cholesterol level, increased plasma triglycerides and a higher risk of developing obesity as

well as type 2 diabetes which are found in T1128C carriers (Karvonen et al., 1998; Pesonen,

2008).

3.4 Conclusion

In conclusion my experiments demonstrate that the L7P SNP does not alter the

trafficking pattern of proNPY but leads to an increase in prohormone packaging into dense core

granules and an elevation in NPY peptide secretion. The results also emphasize that functional

SNPs can have readily detectable effects at the level of single granules. Most of this work has

been published in the Journal of Neuroscience (Mitchell *, Wang *, Ramamoorthy and Whim

(2008) 28: 14428-34; * co-first author).

Chapter 4

Acute Stress Increases the Levels of Adrenal Neuropeptide Y

4.1 Introduction

Every individual experiences stressful life events and the ability to cope with stress is

extremely important to prevent harmful outcomes. The beneficial aspect of the acute stress

response is that it triggers an adaptive response which allows the organism to cope with the

stressor. The harmful aspect can be evident during exposure to a chronic stress which can elicit a

pathological response (McEwen, 1998; Sabban et al., 2006).

People with poor stress coping ability are highly susceptible to numerous diseases such

as depression and other psychiatric disorders (Feder et al., 2009). Chronic stress also leads to the

development of cardiovascular disease (Kuo et al., 2007a) and increases the risks of immune

dysfunction and cancer (Johnstone and Baylin, 2010; McEwen, 1998; Wong, 2006). In addition

chronic stress is associated with an increased susceptibility to obesity and drug addiction (Kuo et

al., 2007b; Ungless et al., 2010).

Exposure to either an acute or chronic stressor results in a series of coordinated responses

consisting of changes in behavior, neuronal plasticity, neuroendocrine functions and secretion of

hormones (Lupien et al., 2009; Roozendaal et al., 2009; Van de Kar and Blair, 1999). In the CNS

the stress response takes place in many brain regions including the hippocampus (Pittenger and

Duman, 2008), hypothalamus (Hewitt et al., 2009), amygdala (Knoll et al., 2011), prefrontal

cortex (Yuen et al., 2011), ventral tegmental area (Krishnan et al., 2007) and cerebellum (Liu et

al., 2010). In the PNS the stress response involves two major pathways, both of which are

initiated by hypothalamic activity (Axelrod and Reisine, 1984; Sapolsky et al., 2000; Wong,

57

2006). One is the “hormonal pathway” in which stress activates the hypothalamus-pituitary

gland-adrenal cortex axis (known as the HPA axis) and leads to a release of corticotropin-

releasing hormone (CRH) from the hypothalamus. CRH induces secretion of adrenocorticotropin

hormone (ACTH) from the pituitary gland. The release of ACTH stimulates the adrenal cortex to

secrete glucocorticoids and then glucocorticoids participate in a negative feedback loop, which

inhibits the activity of the hypothalamus and pituitary to prevent the over-activation of the HPA

axis (Figure 4-1 A) (Axelrod and Reisine, 1984; Sapolsky et al., 2000). The other peripheral

stress pathway is the “neuronal pathway” characterized by the activation of the sympatho-adrenal

medullary system. Descending input from the hypothalamus activates spinal preganglionic

neurons. Some of these neurons project to their targets via splanchnic nerves. Acetylcholine,

which is the primary neurotransmitter in the splanchnic nerves, is released, depolarizing the

chromaffin cells in the adrenal medulla leading to the release of the catecholamines (mainly

epinephrine), which alters heart rate and blood pressure (Figure 4-1 B) (Axelrod and Reisine,

1984; Wong, 2006).

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Figure 4-1.The two major peripheral stress pathways.

(A). The hormonal pathway; also known as the HPA axis. (B). The neuronal pathway; the

sympatho-adenal medullary system.

The stress-induced release of hormones including ACTH from the pituitary gland,

glucocorticoids from the adrenal cortex, epinephrine from the adrenal medulla and

norepinephrine from the sympathetic nerves (with a minor contribution from the adrenal medulla)

helps to achieve stability which prevents the development of a pathological response to stress

(Axelrod and Reisine, 1984).

Each adrenal gland consists of a cortex and a medulla, both of which are involved in the

stress response. Therefore the adrenal glands are part of the mechanisms that allow the organism

to cope with stress (Carrasco and Van de Kar, 2003). The functional unit of the adrenal medulla is

the chromaffin cell, a modified postganglionic neuron which synthesizes and secretes hormones,

not only the catecholamines, but also NPY (Varndell et al., 1984).

Expression of adrenal NPY mRNA is elevated by various stressors including cold and

immobilization (Hiremagalur et al., 1994; Hiremagalur and Sabban, 1995; Raghuraman et al.,

2011). The levels of adrenal NPY mRNA are increased by a relatively short period of

immobilization and the elevation is abolished by the transcriptional inhibitor actinomycin D.

Furthermore this stress-induced rise of NPY mRNA expression requires splanchnic innervation

suggesting that the neuronal activation of chromaffin cells is essential for the immobilization-

induced increase of NPY transcription (Hiremagalur et al., 1994).

Although the adrenal mRNA levels of NPY are increased by several stressors, it is still

not clear whether acute stress increases the levels of the NPY peptide in chromaffin cells and if

the response is seen in all chromaffin cells.

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Here I investigate these questions by exposing mice to an acute stressor, the cold water

forced swim test (cold FST) or fox urine odor. I find that the cold FST and exposure to the odor

of fox urine leads to an increase in NPY immunoreactivity (NPY-ir) in chromaffin cells. Stress

increases the number of chromaffin cells that express relatively high levels of NPY peptide and

decreases the number of cells that contain relatively low levels of NPY-ir. The increase in NPY

synthesis in chromaffin cells also involves an increase in NPY transcription. In addition the cold

FST increases the levels of NPY in adrenal slices and the increase has reversed one week later.

4.2 Results

4.2.1 The cold FST activated the HPA axis and induced activity in chromaffin cells

The cold FST is a common stressor used in investigating the effect of acute stress and

was shown to enhance synaptic transmission in dopamine neurons in the CNS (Huber et al., 2001;

Saal et al., 2003). However it has not been used widely in the context of the peripheral stress

response. To examine its validity as a peripheral stressor, mice were exposed to the cold FST for

5-6 minutes and decapitated thirty minutes later (Figure 4-2 A). Blood was collected for

measurement of the levels of plasma corticosterone, the adrenal component of the HPA axis.

Plasma corticosterone levels were significantly higher in the stressed animals compared to the

matched controls (Figure 4-2 B) indicating that the cold FST led to an activation of the HPA axis.

I then asked if the cold FST activated the neuronal branch of the stress response in

addition to the hormonal axis. c-Fos is an immediate early gene whose expression is an indirect

marker of neuronal activity (Jhou et al., 2009; Lobo et al., 2010). C-Fos-ir in adrenal slices was

examined 60 minutes after the cold FST and compared to the matched controls (Figure 4-3 A).

The number of c-Fos-ir cells was significantly increased in slices of the adrenal medulla after

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stress (Figure 4-3 B and C) indicating that the cold FST elevated the electrical activity of

chromaffin cells.

Thus the cold FST activated both the HPA axis and the neuronal branches of the stress

response.

4.2.2 GFP is expressed in the hypothalamus, hippocampus and adrenal medulla in NPY

(GFP) transgenic mice

I obtained a NPY-hrGFP transgenic mouse line that was generated using a large bacterial

artificial chromosome (BAC) containing the NPY promoter and upstream regulatory elements

that drives the expression of Renilla GFP. These mice were shown to express GFP in most of the

known NPY neurons in the brain (van den Pol et al., 2009). In these mice I confirmed that GFP

was richly distributed in the hypothalamus and the hippocampus (Figure 4-4 A and B). I found

that GFP was also expressed in the adrenal medulla but not in the adrenal cortex suggesting that

NPY in the adrenal glands was only made by chromaffin cells (Figure 4-4 C). Moreover GFP was

found in all chromaffin cells.

4.2.3 An NPY antibody specifically recognizes NPY in chromaffin cells

To test the specificity of an NPY antibody that would be used in later experiments, I

stained chromaffin cell cultures from wild type and NPY knockout mice with the NPY antibody.

Chromaffin cells from the wild type animal were stained with the NPY antibody (Figure 4-5 A)

In contrast chromaffin cells from the knockout animal were not stained with the NPY antibody

(Figure 4-5 B). The lack of NPY-ir in chromaffin cells from the NPY knockout mouse indicates

that the NPY antibody I used specifically recognizes NPY in the adrenal medulla.

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4.2.4 The cold FST led to an up-regulation of NPY-immunoreactivity in chromaffin cells in

vitro

To determine whether the cold FST regulated the levels of adrenal NPY in chromaffin

cells, animals were exposed to the cold FST for 5 to 6 minutes. After 24 hours the chromaffin

cells were dissociated from stressed and matched control animals (Figure 4-6 A). Quantification

of the levels of NPY-ir in single chromaffin cells from stressed and littermate control mice

indicated that stress increased NPY-ir (Figure 4-6 B and C). The frequency histograms of the

levels of NPY-ir in chromaffin cells from both matched control and stressed mice was better

fitted with two Gaussian distributions compared to one (Figure 4-6 D). This indicates that there

are two sub-populations of chromaffin cells, one with relatively high levels of NPY-ir and the

other with lower levels of NPY-ir. The distributions of NPY-ir in chromaffin cells showed that

the cold FST increased the number of cells expressing high levels of NPY but decreased the

proportion of cells with low NPY-ir compared with the matched controls (Figure 4-6 D). The

rightward shift in the cumulative distribution of intensities from the cold FST-exposed animals

suggested that chromaffin cells from stressed mice contained higher levels of NPY (Figure 4-6 E).

My findings suggest that the cold FST-induced stress increases the levels of NPY-ir due to a shift

between two sub-populations of chromaffin cells.

The levels of NPY-ir in chromaffin cells dissociated from a mouse which was sacrificed

30 minutes after the cold FST was not different from the matched control (Appendix A). This

result indicated that the stress-induced increase in the levels of NPY-ir in chromaffin cells

required an extended period of time to become evident.

Previous studies have shown that the mRNA levels of NPY in whole adrenal glands were

altered by immobilization or cold (Hiremagalur et al., 1994; Hiremagalur and Sabban, 1995;

Raghuraman et al., 2011). The present work shows that the levels of NPY rise in single

chromaffin cells.

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4.2.5 Brief exposure to fox urine also led to an up-regulation of NPY-immunoreactivity in

chromaffin cells in vitro

To investigate whether the effect of acute stress on the levels of adrenal NPY was

universal or restricted to one specific stressor, I exposed mice to a different stressor, fox urine

odor for 5 minutes after which the animals were returned to their home cage (Figure 4-7 A). Fox

urine exposure induces an innate fear in mice as indicated by a freezing behavior (Hu et al., 2007;

Liu et al., 2010). Sixteen to twenty four hours after the fox urine exposure, I measured the levels

of NPY-ir in single chromaffin cells dissociated from stressed and matched control animals.

Similar to the cold FST-induced stress, the fox urine exposure also increased NPY-ir in

chromaffin cells (Figure 4-7 B, C and E). In a similar fashion to the cold FST, the fox urine

exposure led to a sub-population shift of NPY-ir in chromaffin cells (Figure 4-7 D).

Thus two different types of acute stressor both increased NPY-ir in chromaffin cells in a

similar manner. This suggests that acute stress leads to a coordinated up-regulation of the levels

of adrenal NPY.

4.2.6 The cold FST increased NPY-ir measured in adrenal slices

Having found that acute stress increased NPY-ir in chromaffin cell cultures in vitro, I

next examined the levels of NPY-ir in adrenal slices from cold FST- exposed mice and matched

controls. The advantage of this approach is that the structure of the gland is preserved. Frozen

sections were made from control and stressed animals 24 hours after the cold FST, and stained

with the NPY antibody. The cold FST significantly increased the levels of NPY-ir in the adrenal

medulla (Figure 4-8 A and B). Therefore acute stress increases the levels of adrenal NPY in situ

which is consistent with the results from chromaffin cells in vitro.

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The levels of NPY-ir in adrenal slices from a mouse which was sacrificed 30 minutes

after the cold FST was not different from the matched control (Appendix A). This is consistent

with the result that NPY-ir in chromaffin cells in vitro was not changed 30 minutes after the cold

FST (section 4.2.4).

4.2.7 The cold FST-induced increase of NPY-ir in adrenal slices returned to baseline one

week after stress

Acute stress is believed to be part of an adaptive mechanism that enables an organism to

cope with stress, in contrast to chronic stress which may cause depression. I next asked whether

the stress-induced increase of NPY was reversible. This is important because over-activation of

the adrenal system in the stress response can be pathological. Animals were exposed to the cold

FST then sacrificed either 24 hours or one week later. Matched control animals were treated

identically except they were not exposed to stress (Figure 4-9 A). I measured and compared NPY-

ir in the adrenal medulla from these three groups of animals. NPY-ir in the adrenal medulla was

significantly higher at 24 hours after the cold FST but returned to control values by one week

after acute stress (Figure 4-9 B and C). The return of adrenal NPY-ir back to baseline after stress

may imply that a negative feedback regulatory pathway is involved in the adrenal response to

stress.

4.2.8 The cold FST increased GFP expression in NPY (GFP) transgenic mice

Since stress increased the adrenal levels of the NPY peptide, I then investigated whether

stress increased the levels of adrenal NPY via a process that involved an increase of NPY gene

transcription or a reduction of NPY secretion (which would lead to increased levels of

intracellular NPY). To test these possibilities I used an NPY (GFP) transgenic mouse, in which

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GFP is linked to the NPY promoter and the NPY signal sequence is interrupted. These BAC NPY

(GFP) transgenic mice still make endogenous NPY (van den Pol et al., 2009). The NPY (GFP)

mice were exposed to the cold FST and 24 hours later chromaffin cells were dissociated from

stressed mice and matched controls. I measured the expression of the reporter gene by the

fluorescence levels of GFP. GFP fluorescence was significantly higher in chromaffin cells from

stressed animals compared to matched controls (Figure 4-10 A, B and D). The frequency

histograms of GFP fluorescence in chromaffin cells from both matched control and stressed mice

was fitted with two Gaussian distributions better than one (Figure 4-10 C). Comparison of the

frequency histograms of GFP fluorescence from control and stressed animals showed a sub-

population shift in the GFP fluorescence after stress in a similar manner to that seen with NPY-ir

(Figure 4-10 C).

The increased GFP expression after stress suggested that the cold FST elevated NPY

promoter activity in chromaffin cells since the GFP expression was driven by the NPY promoter.

Unlike NPY, GFP is a non-secreted protein so the increase of GFP expression after stress was due

to an increased synthesis rather than reduced secretion (although I still cannot rule out the

possibility that a reduced NPY secretion contributes to the increase of the adrenal peptide). The

data from the NPY (GFP) animals suggested that stress increased the levels of adrenal NPY by a

process that involved an increase of gene transcription and peptide production.

4.2.9 Adrenal NPY mRNA expression was increased 3 hours after the cold FST

Since adrenal NPY promoter activity was shown to increase in the NPY (GFP) animals, I

then measured the levels of NPY mRNA in the adrenal medulla from the wild type animals by

semi-quantitative RT-PCR. The results indicated an increase in NPY mRNA expression 3 hours

after the cold FST (Figure 4-11 A and B). This supports the data from the NPY (GFP) transgenic

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mice that the NPY promoter activity was increased after stress. Therefore the cold FST-induced

increase of adrenal NPY is likely to involve transcriptional regulation.

4.2.10 Pancreatic polypeptide and peptide YY mRNA expression in the adrenal medulla

after the cold FST

The NPY family of peptides includes two other members, pancreatic polypeptide (PP)

and peptide YY (PYY), which are primarily found in the pancreas and intestine, respectively

(Ekblad and Sundler, 2002). No PYY-ir or mRNA has been reported in the rat adrenal medulla.

However PYY-ir was shown to be present in the cat adrenal medulla (Ekblad and Sundler, 2002;

Pieribone et al., 1992). PP-like immunoreactivity in the rat adrenal medulla was reported but the

antibody used was not well characterized (Pieribone et al., 1992; Vaillant and Taylor, 1981).

Later studies using a well-defined antibody and a radioimmunoassay failed to detect the

expression of PP in the rat adrenal medulla (Miyazaki and Funakoshi, 1988; Pieribone et al.,

1992).

To test whether PP and PYY are expressed in the mouse adrenal medulla and whether

stress can affect their expression, I utilized RT-PCR to examine the mRNA levels of PP and PYY.

mRNA encoding PP and PYY was present in mouse pancreatic islets validating the primers that I

used. Insulin was used as a positive control for islet tissue (Figure 4-12 A). Amplification of NPY

in the adrenal medulla indicated the successful extraction of mRNA and generation of cDNA.

Neither PP nor PYY expression was detected in the adrenal medulla of control mice in three

independent experiments (Figure 4-12 B and C). No PP expression was present in the animals 24

hours after the cold FST (Figure 4-12 B and C). However 24 hours after stress, in one experiment

PYY mRNA was detected in the adrenal medulla whereas the other two experiments did not

reveal the amplification of PYY (Figure 4-12 B and C). The apparent variability in adrenal PYY

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expression in the stressed mice may depend on the different individuals or the intensity of the

stress. The result implies that PYY might be expressed in the mouse adrenal medulla, perhaps

under extremely stressful circumstances.

In conclusion, among the NPY family of peptides, only NPY was consistently expressed

in the mouse adrenal medulla and only this member of the Y peptide family was increased by

exposure to different stressors.

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Figure 4-2. The cold FST activated the HPA axis.

(A). Stress paradigm. P21-P23 male littermates were used in each individual experiment. (B).

Plasma corticosterone levels increased 30 minutes after the cold FST (mean ± SEM, n = 5). **p <

0.01, Student’s paired t test. The open black circles indicate the value from each individual

animal. The black lines link the matched control and stressed animals from each individual

experiment.

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Figure 4-3. The cold FST activated the neuronal branch of the stress response.

(A). Stress paradigm. P21-P23 male littermates were used in each individual experiment. (B).

Adrenal glands stained for c-Fos from control and stressed animals. The number of c-Fos-ir cells

in the medulla (within the white dashed lines) were quantified using multiple areas of interest

(white circles). The enlarged inset represented the region that was outlined by the white square.

Scale bars 100 μm. (C). Group data showing that stress increased the number of c-Fos-ir cells in

the medulla (mean ± SEM, n = 3). *p < 0.05; Student’s paired t test. Seven to ten sections were

used to measure c-Fos-ir from each animal. The open black circles indicate the value from each

individual animal. The black lines link the matched control and stressed animals from each

individual experiment.

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Figure 4-4. NPY (GFP)-expressing neurons and neuroendocrine cells in the nervous system.

(A). NPY (GFP) neurons in the mouse hippocampus. (B). NPY (GFP) neurons in the mouse

hypothalamus. (C). NPY (GFP) is expressed in the mouse adrenal medulla.

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Figure 4-5. NPY-ir in chromaffin cells from wild type and NPY knockout mice.

(A). Dissociated chromaffin cells from a wild type animal were stained with an NPY antibody.

(B). Chromaffin cells from an NPY knockout animal did not exhibit any NPY-ir when stained

with the NPY antibody. The enlarged inset is a bright field image of chromaffin cells. The white

dashed circles outline chromaffin cells in the NPY knockout animal. Scale bar 5 μm.

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Figure 4-6. The cold FST increased the level of NPY-ir in chromaffin cells in vitro.

(A). Stress paradigm. P21-P23 male littermates were used in six of the seven independent

experiments, and one paired group were P21 female littermates. (B). Examples of chromaffin

cells from control and stressed animals. Scale bar 10 μm. (C). Group data indicated that NPY-ir is

higher in chromaffin cells from stressed animals compared to matched controls (mean±SEM, n =

7). **p < 0.01; Student’s paired t test. Seventy cells were used from each animal to measure the

levels of NPY-ir. The open black circles indicate the value from each individual animal. The

black lines link the matched control and stressed animals from each individual experiment. (D).

Representative frequency histogram from either matched control or stressed mice fitted with two

Gaussian distributions (red dashed curve vs. navy blue dashed curve). The representative

frequency histogram showed that stress resulted in a decrease in the proportion of low NPY-ir

cells and an increase in the proportion of high NPY-ir cells. (E). The cumulative distributions

indicated chromaffin cells from stressed animals contained higher levels of NPY. ***p < 0.001;

Kolmogorov-Smirnov test.

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Figure 4-7. Brief exposure to fox urine increased the level of NPY-ir in chromaffin cells in

vitro.

(A). Stress paradigm. P19-P21 male or female littermates were used. (B). Chromaffin cells from

control and stressed animals. Scale bar 10 μm. (C). Group data indicated that NPY-ir is higher in

chromaffin cells from stressed animals compared to matched controls (mean ± SEM, n = 6). *p <

0.05; Student’s paired t test. Sixty to seventy cells were used from each animal to measure NPY-

ir. The open black circles indicate the value from each individual animal. The black lines link the

matched control and stressed animals from each individual experiment. (D). A representative

frequency histogram from either matched control or stressed mice fitted with two Gaussian

distributions (red dashed curve vs. navy blue dashed curve). The representative frequency

histogram showed that stress led to a decrease in the proportion of low NPY-ir cells and an

increase in the proportion of high NPY-ir cells. (E). The cumulative distributions indicated

chromaffin cells from stressed animals contained higher levels of NPY. ***p < 0.001;

Kolmogorov-Smirnov test.

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Figure 4-8. The cold FST increased the level of adrenal NPY-ir in situ.

(A). Adrenal sections from a control animal and 24 hours after the cold FST. Scale bars 100 μm.

(B). Group data showing that NPY-ir was significantly higher in the adrenal medulla from

stressed animals compared to the matched controls. NPY-ir was normalized to the values of

controls (mean ± SEM, n = 3; raw data: 94.15 ± 23.42 vs. 173.75 ± 34.01 arbitrary units). *p <

0.05; Student’s matched one sample t test. P21-P23 male littermates were used in each individual

experiment. Seven to thirteen sections were used to measure NPY-ir from each animal. The open

black circles indicate the value from each individual animal. The black lines link the matched

control and stressed animals from each individual experiment.

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Figure 4-9. The cold FST reversibly increased the level of adrenal NPY-ir in situ.

(A). Stress paradigm. P21-P23 male littermates were used in two of the three independent

experiments, and one paired group were P21 female littermates. (B). Adrenal sections from a

control animal, 24 hrs and 1 week after the FST. Scale bars 100 μm. (C). Group data showed that

NPY-ir was significantly higher in the adrenal medulla at 24 hrs after the cold FST but declined

to control values by 1 week after the FST (mean ± SEM, n = 3). *p < 0.05; **p < 0.01; one-way

ANOVA (post-hoc Tukey’s paired comparison; F (2, 6) = 16.31). Eight to ten sections were used

to measure the levels of NPY-ir from each animal. The open black circles indicate the value from

each individual animal. The black lines link the matched control and stressed animals from each

individual experiment.

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Figure 4-10. The cold FST increased GFP expression in chromaffin cells from NPY (GFP)

mice.

(A). GFP expression in chromaffin cells from a control and stressed animal. Scale bar 10 μm. (B).

Group data indicated that GFP expression was significantly higher in chromaffin cells from the

stressed animals compared to the matched controls (mean±SEM, n = 3). **p < 0.01; Student’s

paired t test. P21-P22 male littermates were used in each individual experiment. Ninety cells were

used from each animal to measure GFP fluorescence. The open black circles indicate the value

from each individual animal. The black lines link the matched control and stressed animals from

each individual experiment. (C). A representative frequency histogram from either matched

control or stressed mice fitted with two Gaussian distributions (red dashed curve vs. navy blue

dashed curve). The representative frequency histogram showed that stress led to a decrease in the

proportion of low GFP-expressing cells and an increase in the proportion of high GFP-expressing

cells. (D). Cumulative distributions indicated that chromaffin cells from the stressed animals

contained higher levels of GFP. ***p < 0.001; Kolmogorov-Smirnov test.

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Figure 4-11. The adrenal levels of NPY mRNA increased 3 hours after the cold FST.

(A). An example of a RT-PCR experiment for NPY and actin from a control animal and 3 hours

after the cold FST. The negative controls were RT reactions without reverse transcriptase. (B).

Group data indicated that the level of NPY mRNA was significantly higher in the stressed

animals. Actin expression was used as an internal control. Results from the stressed animals were

normalized to matched controls (n = 3). *p < 0.05; Student’s matched one sample t test. P21-P23

male mice were used. The open black circles indicate the value from each individual animal. The

black lines link the matched control and stressed animals from each individual experiment.

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Figure 4-12. Expression of PP, PYY and NPY mRNA in the adrenal medulla.

(A). RT-PCR analysis showed the expression of PP (196 bp), PYY (260 bp) and insulin (241 bp)

in pancreatic islets. (B). An example of RT-PCR for PP, PYY and NPY (288 bp) from the adrenal

medulla in one group of control and stressed animals. (C). An example of RT-PCR of PP, PYY

and NPY from the adrenal medulla in a second group of control and stressed animals. The

negative controls were RT reactions without reverse transcriptase. P21 male littermates were used.

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4.3 Discussion

4.3.1 Stress increases adrenal NPY transcription and peptide synthesis.

Various stressors such as cold and immobilization are known to elevate NPY gene

expression in the adrenal medulla (Hiremagalur et al., 1994; Hiremagalur and Sabban, 1995;

Raghuraman et al., 2011). The stress-induced increase of adrenal NPY is transcription-dependent

since the transcriptional inhibitor actinomycin D abolished the effect of stress on adrenal NPY

gene expression (Hiremagalur et al., 1994). In my study I also found that the cold FST-induced

stress increased NPY promoter activity in chromaffin cells.

Although the levels of adrenal NPY mRNA were up-regulated by stress, the effect of

stress on NPY peptide synthesis and how it is mediated at the single cell level is not clear. My

study demonstrates a stress-induced increase of NPY peptide synthesis in chromaffin cells. Since

I am not able to directly measure the secretion of endogenous NPY from single chromaffin cells,

I compared GFP expression in control and stressed NPY (GFP) transgenic mice as a way to

determine whether stress increased NPY synthesis. The stress-induced increase of GFP

expression in chromaffin cells from the NPY (GFP) mice confirmed that the increased levels of

adrenal NPY 24 hours after stress involved an up-regulation of NPY production rather than a

reduction in secretion. In the previous chapter (section 3.2.7), I showed that an increased level of

NPY biosynthesis likely leads to an elevation of evoked NPY peptide release. Thus the stress-

induced long-term up-regulation of adrenal NPY synthesis may lead to an increase of NPY

secretion and have profound effects on metabolic and cardiovascular regulation.

I also investigated the cellular actions involved in the response of the adrenal medulla to

stress and found a shift in the expression of NPY between two sub-populations of chromaffin

cells after stress. Chromaffin cells from control animals express both low and high levels of NPY.

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Stress increases the number of cells that express high levels of NPY but does not up-regulate the

NPY levels in all of the chromaffin cells. I speculate a possible mechanism for this stress-induced

sub-population shift may involve the following: (1) some cells that contain a high level of NPY

have reached a threshold and NPY cannot be further increased; (2) a portion of NPY-containing

cells that express low levels of NPY lack the components that are needed to sense the signaling

pathways triggered by stress; (3) other cells with low levels of NPY can be activated by stress and

these cells switch between expressing low and high levels of NPY during the stress response. It is

known that there are sub-populations in chromaffin cells: for example the epinephrine-containing

vs. norepinephrine-containing cells (Cahill et al., 1996). Since all mouse chromaffin cells express

NPY, the different sub-populations in the levels of NPY may function in regulating the

expression of other hormones in chromaffin cells.

4.3.2 The mechanism that is involved in the stress-induced increase of adrenal NPY.

The immobilization-induced increase of adrenal NPY requires splanchnic innervation

which is the neuronal input into the adrenal medulla (Hiremagalur et al., 1994). The evidence for

the necessary involvement of the sympathetic innervation in the stress-induced increase of NPY

mRNA is that either denervation by splanchnicectomy or injection of the nicotinic receptor

antagonist chlorisondamine blocks the immobilization-induced elevation of NPY mRNA level

(Hiremagalur et al., 1994; Hiremagalur et al., 1995).

4.3.3 The transcriptional factors that are involved in response of the adrenal medulla to

stress.

Acute stress elicits a global change of gene expression and the greatest number of

changes in the adrenal medulla are observed within transcription factors (Liu et al., 2008). c-Fos

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is not only an indirect marker of neuronal activation, but also a transcriptional factor which is

activated by stress (Figure 4-2 A and B)(Kovacs, 1998). The NPY promoter region contains an

AP1 binding site motif which is potentially induced by c-Fos activation (Titolo et al., 2008).

However direct binding of c-Fos with the NPY promoter still lacks experimental evidence.

Delta FosB is a truncated splice isoform of FosB, another Fos family member which

participates in forming the transcriptional factor complex AP1 (Nestler, 2008). Delta FosB in the

nucleus accumbens, an important brain rewarding circuit was shown to mediate resilience in the

chronic social defeat stress response. A genome-wide analysis of gene expression in the nucleus

accumbens indicated an increase of NPY gene expression in the mice with an overexpression of

delta FosB compared to the controls (Vialou et al., 2010). Delta FosB expression in the adrenal

medulla was significantly increased by repeated immobilization stress (Nankova et al., 2000).

Overexpression of delta FosB in PC12 cells (a cell line derived from a tumor of the rat adrenal

medulla) resulted in an increase of tyrosine hydroxylase promoter (containing an AP1- like

binding motif) activity (Nankova et al., 2000). Therefore delta FosB may also potentiate adrenal

NPY transcription.

Besides the Fos family of proteins, the adrenal cAMP response element-binding protein

(CREB) is significantly activated by stress and MAPK-ERK1/2 signaling, an upstream regulator

of CREB is also induced (Sabban et al., 2006). There is a half-CRE site in the NPY promoter

region and CREB directly binds to the -118 to +22 bp region of the NPY promoter in the N-38

neuronal cell line as shown by a chromatin immunoprecipitation assay (Titolo et al., 2008).

Since the immobilization stress-induced increase in adrenal NPY transcription requires

neuronal innervation of chromaffin cells (Hiremagalur et al., 1994; Hiremagalur et al., 1995), c-

Fos which is activated during neuronal firing is a good candidate to mediate the effect of stress on

an increase of adrenal NPY transcription (Labiner et al., 1993). In addition the activation of

chromaffin cells during the stress response causes an elevation of Ca2+

influx. Therefore the

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pathways activated by Ca2+

influx could account for the stress-induced increase of NPY and this

may involve CREB activation (Labiner et al., 1993).

Although the transcriptional factors c-Fos (section 4.2.1), delta Fos B (Nankova et al.,

2000) and CREB (Sabban et al., 2006) in the adrenal medulla are activated by stress and

potentially induce NPY gene expression, there is no direct evidence to link them with the stress-

induced increase of adrenal NPY gene expression. Therefore the molecular mechanisms and

signaling molecules involved in the stress-induced elevation of adrenal NPY transcription need to

be determined in future experiments.

4.3.4 Adrenal epinephrine in the stress response.

The activation of chromaffin cells after stress leads to an increase of catecholamine

release within seconds (Axelrod and Reisine, 1984). The elevation of plasma catecholamines

might be part of the pathway that leads to the increase of adrenal NPY transcription. Splanchnic

denervation which blocks catecholamine release abolishes the stress-induced adrenal NPY up-

regulation (Hiremagalur et al., 1994; Hiremagalur et al., 1995).

In preliminary experiments I found that injection of epinephrine increased adrenal NPY-

ir (Appendix B) and direct incubation of adrenal slices with epinephrine also elevated NPY-ir

(Appendix B). This data may indicate that the stress-induced increase in plasma catecholamines is

an upstream signaling event which activates adrenal NPY gene expression. Both alpha and beta

adrenergic receptors are expressed in the adrenal medulla and they are all G protein coupled

receptors (Cesetti et al., 2003; Kleppisch et al., 1992). A regulatory mechanism involving adrenal

catecholamine secretion has been described that acts via autocrine feedback inhibition of α2

adrenergic receptors in chromaffin cells (Lymperopoulos et al., 2007). Since the activation of beta

adrenergic receptor leads to an increase of cAMP (Cesetti et al., 2003), adrenal catecholamine

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release may exert autocrine activation through beta adrenergic receptors to induce the activation

of CREB thereby regulating adrenal NPY gene expression.

4.3.5 Stress-induced secretion and synthesis coupling

The metabolic stress that is triggered by insulin-induced hypoglycemia stimulates the

splanchnic nerve and catecholamine release from the adrenal medulla leading to a depletion of

catecholamines in chromaffin cells (Laslop et al., 1989). The increased plasma catecholamines

ensure that a compensatory glycogenolysis and gluconeogenesis takes place to maintain glucose

homeostasis. The induction of adrenal tyrosine hydroxylase during an insulin challenge enables a

refilling of the depleted pool of catecholamines in chromaffin cells, which is important for

maintaining a continual secretion of catecholamines and thus regulating the levels of plasma

glucose (Hamelink et al., 2002). In pituitary adenylate cyclase-activating polypeptide (PACAP)

knockout mice, there is a failure to increase adrenal catecholamine synthesis during an insulin

challenge. These animals showed a more long-lasting insulin-induced hypoglycemia and an

insulin dose-related lethality compared to wild type animals (Hamelink et al., 2002). These results

suggest an important role for the coupling of secretion and biosynthesis of catecholamine

hormones in response to stress.

Stress stimulates the secretion of NPY from sympathetic neurons and chromaffin cells

(Kuo et al., 2007a). In my experiments I showed that NPY synthesis in chromaffin cells was up-

regulated by stress. The coupling of the stress-induced increase of NPY release and synthesis in

chromaffin cells may be a survival mechanism that allows the organism to maintain physiological

homeostasis.

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4.4 Conclusion

In conclusion my experiments showed that acute stress increased the levels of the NPY

peptide in the adrenal medulla and that this is due to an increased number of cells expressing high

levels of NPY and a reduced number of cells containing low levels of NPY. The increase of the

NPY peptide involved an up-regulation of NPY gene transcription and the increase reversed one

week after the stress.

Chapter 5

NPY Inhibits the Neuronal Branch of the Stress Response

5.1 Introduction

In the previous chapter I described how the stress response is regulated by two major

pathways which are the hormonal pathway (the “HPA axis”) and the neuronal pathway (the

“sympathetic-adrenal medullary system”). Activation of these two pathways leads to the release

of the hormones cortisol (corticosterone in rodents) from the adrenal cortex and the

catecholamines from the adrenal medulla (Axelrod and Reisine, 1984; Sapolsky et al., 2000;

Wong, 2006).

Adrenal cortisol has many important metabolic actions such as promoting hepatic

gluconeogenesis and lipogenesis, and inhibiting both acute and chronic inflammation (Heitzer et

al., 2007; Landys et al., 2006). Adrenal catecholamines are essential during the fight-or-flight

response altering blood pressure, increasing hepatic gluconeogenesis, elevating thermogenesis

and enhancing lipolysis in fat cells (Bachman et al., 2002; Keys and Koch, 2004; Song et al.,

2010; Zhang et al., 2010a).

The catecholamines are synthesized from the precursor amino acid tyrosine. The enzyme

tyrosine hydroxylase (TH) converts tyrosine to dihydroxyphenylalanine (DOPA) and then DOPA

is converted to dopamine (a catecholamine) by aromatic amino acid decarboxylase (DDC).

Dopamine-β-hydroxylase (DβH) catalyzes the generation of norepinephrine (NE, another

catecholamine), which can be converted to epinephrine (Epi, a third type of catecholamine) by

phenylethanolamine N-methyltransferase (PNMT, Figure 5-1). NE and Epi are the

catecholamines found in chromaffin cells. Since TH is the first and rate-limiting enzyme for

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making the catecholamines, it is often used as a marker of catecholamine biosynthesis

(Kvetnansky et al., 2009). A typical response of the adrenal medulla to stress involves not only an

increase in the secretion of catecholamines within seconds but also an induction of TH expression

(Sabban et al., 1995).

Figure 5-1. Catecholamine biosynthetic pathway.

Tyrosine is the precursor of the catecholamines. TH is the rate-limiting enzyme. Dopamine, NE

and Epi are different types of catecholamines but only NE and Epi are found in chromaffin cells.

NPY another important hormone produced and secreted by the adrenal medulla, is up-

regulated by stress (Hiremagalur et al., 1994). However the physiological functions of NPY in

modulating adrenal activity during the stress response are still unknown. Here I ask whether NPY

regulates the adrenal components in either branch of the stress response.

Previous in vitro studies showed that the catecholamines and NPY were co-released

(Whim, 2006) and most studies reported that NPY stimulated catecholamine secretion (Cavadas

et al., 2002; Cavadas et al., 2001). It was also reported that NPY inhibited TH promoter activity

in the SK-N-MC cell line through NPY Y1 receptors (Cavadas et al., 2006). These in vitro

experiments indicate a strong association between NPY and catecholamines, both of which are

components of the neuronal branch of the stress response.

In this chapter I test the hypothesis that NPY inhibits the expression of adrenal TH thus

repressing the neuronal branch of the stress response. In the following experiments I employed

NPY knockout mice and also pharmacologically blocked the actions of NPY by injecting

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antagonists of NPY receptors into mice. I found that (1). NPY selectively inhibited the neuronal

branch of the stress response by down-regulating the levels of adrenal TH via the Y1 receptor; (2).

NPY also reduced adrenal NPY expression via Y2 receptors.

5.2 Results

5.2.1 The cold FST led to a sustained activation of the HPA axis

In chapter 4.2.1, I showed that the cold FST activated the HPA axis 30 minutes after

stress. Here I measured mouse plasma corticosterone 24 hours after the cold FST. Unexpectedly,

plasma corticosterone was significantly higher in animals 24 hours after the cold FST compared

to control animals suggesting a sustained activation of the HPA axis (Figure 5-2).

The mice I used to exposure to the cold FST were between P24 and P31, which is an

early adolescent period in rodents (Lupien et al., 2009). In rodents brain development occurs

during this time and the HPA axis shows a prolonged activation in response to stress due to the

incomplete maturation of negative-feedback systems (Goldman et al., 1973; Vazquez and Akil,

1993). This may be the reason for the long-lasting effect of the cold FST on plasma

corticosterone levels in these animals.

5.2.2 The cold FST led to a sustained activation of the neuronal pathway

In chapter 4.2.2 the increase of c-Fos expression 1 hour after the cold FST indicated an

activation of the neuronal pathway. In the following experiment I measured the plasma

catecholamine levels 24 hours after the cold FST. The majority of plasma epinephrine comes

from the adrenal medulla whereas most norepinephrine is from sympathetic neurons (Kvetnansky

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et al., 2009). The fold change in plasma levels of the catecholamines showed that plasma

epinephrine was consistently elevated even 24 hours after the cold FST (Figure 5-3) suggesting

that the cold FST induced a sustained activation of chromaffin cells.

Therefore the cold FST led to a maintained activity in both the HPA and the neuronal

axis of the stress response.

5.2.3 Co-localization of NPY and TH in the adrenal medulla

NPY-ir and TH-ir are co-localized in the chromaffin cells (Figure 5-4 A). I then used an

NPY (GFP) transgenic mouse to examine more precisely the expression of NPY and TH in

chromaffin cells. GFP was expressed in all chromaffin cells and was a reliable reporter of NPY

expression since it colocalized with NPY-ir both in vitro and in situ (Figure 5-4 B). TH-ir co-

localized with GFP in all chromaffin cells and adrenal slices indicating that TH and NPY are co-

expressed in all chromaffin cells (Figure 5-4 C).

5.2.4 The cold FST increased the level of PNMT-immunoreactivity in chromaffin cells in

vitro

Chromaffin cells contain two types of catecholamines, either epinephrine or

norepinephrine. PNMT is the enzyme that converts norepinephrine to epinephrine so it is used as

a marker of epinephrine-containing chromaffin cells. Since NPY was found in all mouse

chromaffin cells, NPY is co-expressed with both types of catecholamines in chromaffin cells.

To further investigate the expression of NPY in the two populations of chromaffin cells

(i.e. epinephrine-containing and norepinephrine-containing cells), I measured the levels of both

NPY-ir and PNMT-ir in chromaffin cells from stressed and matched control mice. The levels of

PNMT-ir in chromaffin cells had a continuous distribution (Figure 5-5 A and C). I observed

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chromaffin cells with low and high PNMT-ir (fitting in two Gaussian distributions) in both

control and stressed mice (Figure 5-5 C and D). No correlation was found between the two NPY-

ir sub-populations in chromaffin cells (i.e. low NPY-ir vs. high NPY-ir) and the two PNMT-ir

sub-populations (i.e. low PNMT-ir vs. high PNMT-ir) (Figure 5-5 C).

Stress increased PNMT-ir in single chromaffin cells (Figure 5-5 A, B and E) and more

cells expressed high levels of PNMT-ir and fewer cells contained lower levels of PNMT-irafter

stress (Figure 5-5 D). However it is not clear whether stress increased the number of cells

expressing epinephrine and decrease the number of cells containing norepinephrine, or if stress

only elevated the expression of PNMT in the cells that contained epinephrine.

5.2.5 The cold FST increased tyrosine hydroxylase-immunoreactivity in adrenal slices

To determine the effect of stress on the biosynthesis of the catecholamines in the adrenal

medulla, TH-immunoreactivity (TH-ir) in adrenal slices from stressed mice and matched controls

was examined 24 hours after the cold FST. As mentioned previously TH is the rate-limiting

enzyme for producing the catecholamines, so TH is used as a marker of catecholamine

biosynthesis. I found that the cold FST significantly increased TH-ir in the adrenal medulla

indicating an elevation of catecholamine synthetic capacity in the adrenal medulla in stressed

mice (Figure 5-6 A and B).

5.2.6 The cold FST-induced increase of TH-ir was reversible in adrenal slices

To test whether the increase of TH expression was reversible, I measured TH-ir in mouse

adrenal slices one week after stress compared to matched controls and animals 24 hours after

stress. I found that TH-ir was significantly higher in the adrenal medulla at 24 hours after the cold

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FST but returned towards control values by one week after acute stress (Figure 5-7 A and B). The

increase of adrenal TH-ir reversed 1 week after stress and this is similar to the result that the

stress-induced increased levels of adrenal NPY returned to control values 1 week after stress

(section 4.2.7, Figure 4-9).

5.2.7 The loss of NPY did not affect the stress-induced increase in plasma corticosterone

To investigate the role of NPY in regulating the stress response, I exposed NPY knockout

mice (Erickson et al., 1996) to the cold FST. Twenty-four hours later, plasma corticosterone

levels from the cold FST-exposed mice were significantly higher compared to the control

knockout mice (Figure 5-8), which was a similar response to that seen in wild type animals

(Figure 5-2). These results indicate that the loss of NPY does not obviously affect the

adrenocortical response to stress.

5.2.8 The loss of NPY altered the neuronal branch of the stress response

Although NPY did not affect the stress-induced increase in plasma glucocorticoid, it may

change the neuronal branch of the stress response. Since the catecholamines are one of the key

components in the neuronal pathway, I measured TH-ir in adrenal slices from control and cold

FST-exposed NPY knockout mice. In contrast to the wild type animals, in which the cold FST

increased TH-ir in the adrenal medulla (Figure 5-6 A and B; Figure 5-9 A and B), stress did not

increase TH-ir in the NPY knockout mice. This lack of effect may be due to a basal increase of

TH-ir in the NPY knockout control animals (Figure 5-9 A). Unfortunately the wild type animals

were not littermates of the knockout mice since the NPY knockout mice I obtained are

homozygous knockout with a different genetic background to the C57/BL6 wild type mice. I

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therefore next tested my hypothesis using an alternative strategy as described in the following

experiments.

5.2.9 Expression of NPY Y receptors in the adrenal medulla

To determine which Y receptors are expressed in the adrenal medulla, I used reverse

transcription PCR (RT-PCR) to detect the mRNA expression of the Y receptors. The primers for

Y1, Y2, Y4, Y5 and Y6 were validated using mRNA from whole brain (Figure 5-10 A). RT-PCR

results (using the same amounts of adrenal medulla mRNA and whole brain mRNA) indicated

that Y1, Y2, Y5 and some Y4 were expressed in the adrenal medulla but no y6 (Figure 5-10 B).

Y4 is activated by pancreatic peptide rather than NPY (Michel et al., 1998; Lin et al., 2006), so I

next tested the effects of blocking Y1, Y2 and Y5 by injecting antagonists to these receptors in

the following experiments.

5.2.10 Injection of BIBP3226 a NPY Y1 antagonist was associated with an increase in

adrenal TH-ir

Mice were injected (i.p.) with either saline or BIBP3226 (1mg/kg), a selective NPY Y1

antagonist (Fu et al., 2004) and 15 minutes after the injection, one animal from each injected

group was exposed to the cold FST (Figure 5-11 A). Twenty-four hours later, animals were

decapitated and the level of adrenal TH-ir was examined. I found that TH-ir was significantly

higher in the adrenal medulla from the BIBP3226-injected animals compared to saline-injected

animals (Figure 5-11 B and C).

However the cold FST no longer increased adrenal TH-ir in the saline or BIBP3226-

injected animals. Thus injection per se was sufficiently stressful that it led to an increase in the

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levels of adrenal TH-ir (Figure 5-12). My data suggested that NPY inhibited TH expression via

Y1 receptors, which supports the idea of a basal activation of TH-ir in the NPY knockout mice.

To test whether the effect of blocking Y1 on the increase in adrenal TH expression was

mediated via the actions of NPY, I injected the NPY knockout mice (i.p.) with either saline or

BIBP3226. No difference in the levels of adrenal TH-ir between the saline-injected and

BIBP3226- injected NPY knockout animals was found (Figure 5-13). This result confirmed that

there was a basal activation of TH in the NPY-deficient animals and the inhibitory effect of Y1

on adrenal TH expression was mediated via the actions of NPY. Therefore NPY plays an

important role in down-regulating the levels of adrenal TH via a Y1 receptor.

5.2.11 Injection of BIIE0246 an NPY Y2 antagonist or L152,804, an NPY Y5 antagonist, did

not alter adrenal TH-ir

To examine whether other Y receptors were also involved in regulating adrenal TH, I

blocked the other two Y receptors Y2 and Y5, respectively. I injected animals (i.p.) with either

saline or BIIE0246 (1mg/kg) a selective NPY Y2 antagonist (Dumont et al., 2000) and 15

minutes after the injection, one animal from each injected group was exposed to the cold FST. I

found no difference in the levels of TH-ir in the adrenal medulla between the BIIE0246-injected

animals and saline-injected animals (Figure 5-14 A and B).

I then injected animals (i.p.) with either saline or L152, 804 (10mg/kg) a selective NPY

Y5 antagonist (Kanatani et al., 2000). No difference in the levels of adrenal TH-ir was found in

the L152, 804-injected animals and saline-injected animals (Figure 5-14 C).

Therefore NPY inhibited adrenal TH via NPY Y1 receptors selectively rather than via Y2

or Y5.

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5.2.12 Injection of BIIE0246 was associated with an increase in adrenal NPY-ir

Since NPY is a negative regulator of adrenal TH, a component of the neuronal branch of

the stress response, I next asked whether NPY was also involved in regulating its own expression

since this is another important component of the neuronal pathway. I found that the level of

adrenal NPY-ir from the BIIE0246-injected animals was significantly higher compared to the

saline-injected controls but was not different from the saline-injected cold FST-exposed animals

(Figure 5-15 A and B) indicating that NPY repressed the expression of NPY in the adrenal

medulla via aY2 receptor.

However unlike adrenal TH, the cold FST increased the levels of adrenal NPY-ir in the

saline-injected animals. Thus the injection per se did not lead to an increase of adrenal NPY-ir

levels (Figure 5-16). The different effects of injection on the levels of adrenal TH-ir and NPY-ir

suggest that different stressors regulate NPY and TH expression differently perhaps depending on

the types and/or the intensity of the stress.

5.2.13 Injection of BIBP3226 or L152,804 did not alter adrenal NPY-ir

I then compared the levels of adrenal NPY-ir from the saline-injected and BIBP3226-

injected animals. No difference in the levels of adrenal NPY-ir between the saline-injected and

BIBP0246-injected animals was found (Figure 5-17 A and B).

Then I measured adrenal NPY-ir from the saline-injected and L152, 804-injected animals

and found no difference in the levels of adrenal NPY-ir between the saline-injected and L152,

804-injected animals (Figure 5-17 C). Therefore NPY inhibited the expression of NPY in the

adrenal medulla via Y2 receptors selectively rather than via aY1 or Y5 receptor.

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In conclusion blocking Y1 and Y2 receptors is associated with an increase in the levels of

adrenal TH-ir and NPY-ir respectively, indicating that NPY is a negative regulator of adrenal

functioning by suppressing the synthesis of the catecholamines and NPY. It suggests that the

functional role of NPY may be to prevent a pathological activation of the fight-or-flight response.

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Figure 5-2. The cold FST led to a sustained activation of the HPA axis.

Plasma corticosterone levels were significantly increased 24 hours after the cold FST (mean ±

SEM, n = 8). *p < 0.05; Student’s paired t test. P21-P23 male littermates were used in each

individual experiment. The open black circles indicate the value from each individual animal. The

black lines link the matched control and stressed animals from each individual experiment.

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Figure 5-3. The cold FST led to a sustained activation of the neuronal axis of the stress

response.

(A). Fold change of plasma levels of epinephrine 24 hrs after the FST. *p < 0.05; Student’s

matched one sample t test. P21-P23 male littermates were used in each individual experiment.

Data normalized to control (mean ± SEM, n = 9; raw data: 10.05 ± 2.11 ng/ml vs.13.05 ± 1.00

ng/ml). (B). Fold change of plasma levels of norepinephrine 24 hours after the cold FST. Data

normalized to control (mean ± SEM, n = 9; raw data: 21.38 ± 2.89 ng/ml vs.26.48 ± 4.09 ng/ml).

The open black circles indicate the value from each individual animal. The black lines link the

matched control and stressed animals from each individual experiment.

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Figure 5-4. Co-localization of NPY and TH in chromaffin cells.

(A). Co-staining of NPY-ir and TH-ir in single chromaffin cells. Scale bars 10μm. (B). Co-

localization of GFP and NPY-ir in chromaffin cells from an NPY (GFP) mouse. Upper panel

scale bars 100μm. Lower panel scale bars 10μm. (C). Co-localization of GFP and NPY-ir in

adrenal slices in the NPY (GFP) mouse. Scale bars 100 μm. (D). Co-localization of GFP and TH-

ir in chromaffin cells of the NPY (GFP) mouse. Upper panel scale bars 100μm. Lower panel scale

bars 10μm. (E). Co-localization of GFP and TH-ir in adrenal slices in the NPY (GFP) mouse.

Scale bars 100 μm.

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Figure 5-5. The cold FST increased PNMT-ir in chromaffin cells in vitro.

(A). Examples of chromaffin cells from a control and stressed animal. Scale bar 10 μm. (B). The

levels of PNMT-ir were measured in seventy cells from each animal. The levels of PNMT-ir in

single chromaffin cells were ranked from the lowest value to the highest value and the cells were

numbered from 1 to 70 according to the ranking. NPY-ir (gray dots) and PNMT-ir (blue in

control and red in stress) from the same chromaffin cell were plotted against the number of each

cell. (B). The results of a typical experiment. (C). Group normalized data showed that the level of

PNMT-ir was higher in chromaffin cells from stressed animals compared to matched controls

(mean±SEM, n = 3). *p < 0.05; Student’s matched one sample t test. P21 male littermates were

used in each individual experiment. (D). The representative frequency histogram from either

matched control or stressed mouse fitted in two Gaussian distributions (red dashed curve vs. navy

blue dashed curve). The frequency histogram showed that stress resulted in a decrease in the

proportion of low PNMT-ir cells and an increase in the proportion of intensely PNMT-ir cells. (E).

A plot of the cumulative distributions indicated that chromaffin cells from stressed animals

contained higher levels of PNMT-ir. ***p < 0.001; Kolmogorov-Smirnov test.

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*

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Figure 5-6. The cold FST increased the level of adrenal TH-ir in situ.

(A). Adrenal sections from a control animal and an animal 24 hours after the cold FST. Scale bars

100 μm. (B). Group data showed that the level of TH-ir was significantly higher in the adrenal

medulla from stressed animals compared to matched controls. Data was normalized to control

(mean ± SEM, n = 3; raw data: 105.10 ± 21.49 vs. 230.88 ± 11.48). *p < 0.05; Student’s matched

one sample t test. P21-P23 male littermates were used in each individual experiment. The levels

of TH-ir were measured in eight to ten sections from each animal. The open black circles indicate

the value from each individual animal. The black lines link the matched control and stressed

animals from each individual experiment.

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Figure 5-7. The cold FST reversibly increased the level of adrenal TH-ir in situ.

(A). Adrenal sections from a control animal, 24 hrs and 1 week after the FST. Scale bars 100 μm.

(B). Group data showed that the level of TH-ir was significantly higher in the adrenal medulla at

24 hrs after the cold FST but declined towards control values by 1 week after the FST (mean ±

SEM, n=3). *p < 0.05; **p < 0.01; one-way ANOVA (post-hoc Tukey’s paired comparison;

F (2, 6) = 41.64). P21-P23 male littermates were used in each individual experiment. The levels

of TH-ir were measure in eight to ten sections from each animal. The open black circles indicate

the value from each individual animal. The black lines link the matched control and stressed

animals from each individual experiment.

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Figure 5-8. The cold FST led to a sustained activation of the HPA axis in NPY knockout

mice.

Plasma corticosterone levels were significantly increased 24 hours after the cold FST in the NPY

knockout mice (mean ± SEM, n = 5). *p < 0.05; Student’s paired t test. P21 male littermates were

used in each individual experiment. The open black circles indicate the value from each

individual animal. The black lines link the matched control and stressed animals from each

individual experiment.

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Figure 5-9. The cold FST did not increase the level of adrenal TH-ir in NPY knockout mice.

(A). Adrenal sections from a control animal and 24 hrs after the FST; wild type and NPY

knockout animals are shown. Scale bars 100 μm. (B). TH-ir was significantly higher in the

adrenal medulla from stressed wild type animals compared to matched controls. Data was

normalized to control values (mean ± SEM, n = 3; raw data: 180.00 ± 47.17 vs. 336.85 ± 73.77

arbitrary units). *p < 0.05; Student’s matched one sample t test. However stress did not elevate

TH-ir in the NPY KO animals. Data was normalized to control values (mean ± SEM, n = 3; raw

data: 329.94 ± 73.80 vs. 374.52 ± 65.21 arbitrary units). P21 male wild type littermates or

knockout littermates were used in each individual experiment. The level of TH-ir was measure in

eight to ten sections from each animal. The open black circles indicate the value from each

individual animal. The black lines link the matched control and stressed animals from each

individual experiment.

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Figure 5-10. Expression of NPY Y receptors in the adrenal medulla and whole brain.

(A). RT-PCR experiment examining the expression of Y1, Y2, Y4, Y5, Y6 and NPY in whole

brain. The negative control in the RT-PCR is derived from a reaction using NPY primers but

without reverse transcriptase. (B). RT-PCR experiment examining the expression of Y1, Y2, Y4,

Y5 and NPY mRNA in the mouse adrenal medulla. The same amount of starting mRNA was

used as in (A). The negative control in the RT-PCR is derived from a reaction using NPY primers

but without reverse transcriptase.

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Figure 5-11. Blocking the NPY Y1 receptor was associated with an increase in the level of

adrenal TH-ir.

(A).Stress paradigm. P21 male littermates were used in two of the three independent experiments,

and one paired group were P21 female littermates. (B). Adrenal sections from a control animal

and 24 hours after the cold FST; Mice were injected (i.p.) either with 200 μl saline or BIBP3226

(a Y1 receptor antagonist). Scale bars 100 μm. (C). The level of TH-ir was significantly higher in

the adrenal medulla from the BIBP 3226-injected animals compared to controls. Note that the

cold FST no longer increased TH-ir in the saline or BIBP3226-injected animals. Data are mean ±

SEM, n=3; *p < 0.05, **p < 0.01; one-way ANOVA (post-hoc Tukey’s paired comparison;

F (3, 8) = 6.05). The levels of TH-ir were measured in eight to ten sections from each animal. The

open black circles indicate the value from each individual animal. The black lines link the

matched control and stressed animals from each individual experiment.

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Figure 5-12. Injection per se was sufficiently stressful that it led to an increase in the level of

adrenal TH-ir.

The level of TH-ir was higher in the adrenal medulla from the saline-injected animals

compared to uninjected controls or even to the cold FST-exposed animals (n=2). P21 male

littermates were used in each individual experiment. The levels of TH-ir were measured from

eight to ten sections from each animal. The open black circles indicate the value from each

individual animal. The black lines link the matched control and stressed animals from each

individual experiment.

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Figure 5-13. Blocking the NPY Y1 receptor in NPY knockout mice did not alter the level of

adrenal TH-ir.

The level of TH-ir in the adrenal medulla was not changed when NPY knockout mice were

injected with BIBP3226 compared to saline injection (right).TH-ir was elevated in the cold FST-

exposed wild type animals compared to control (n=1). P21 male littermates were used. The levels

of TH-ir were measured in eight sections from each animal.

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Figure 5-14. Blocking the NPY Y2 or Y5 receptor did not affect the level of adrenal TH-ir.

(A). Adrenal sections from a control animal and 24 hrs after the cold FST; mice were

injected (i.p.) either with 200 μl saline or BIIE0246 (a Y2 receptor antagonist). Scale bars 100 μm.

(B). No difference in the level of TH-ir was detected in the adrenal medulla from the BIIE0246-

injected animals compared to controls. P21 male littermates were used in each individual

experiment. (C). No difference in the level of TH-ir was detected in the adrenal medulla from the

L125,804 (a Y5 receptor antagonist)-injected animals compared to controls. P21 male littermates

were used in two of the three independent experiments, and one paired group were P21 female

littermates. Data are mean ± SEM, n = 3; one-way ANOVA (post-hoc Tukey’s paired comparison;

for (B), F (3, 8) = 0.029; for (C), F (3, 8) = 0.283). The levels of TH-ir were measured from eight

to ten sections from each animal. The open black circles indicate the value from each individual

animal. The black lines link the matched control and stressed animals from each individual

experiment.

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Figure 5-15. Blocking the NPY Y2 receptor was associated with an increase in the level of

NPY-ir in the adrenal medulla.

(A). Adrenal sections from a control animal and 24 hrs after the cold FST; mice were

injected (i.p.) either with saline or BIIE0246. Scale bars 100 μm. (B). BIIE0246-injected animals

had significantly higher level of NPY-ir in the adrenal medulla compared to saline-injected

controls but the same levels as saline-injected cold FST-exposed animals. Note that unlike TH-ir,

the cold FST increased the level of adrenal NPY-ir in the saline or BIIE0246-injected animals.

Data are mean ± SEM, n = 3; *p < 0.05, **p < 0.01; one-way ANOVA (post-hoc Tukey’s paired

comparison; F (3, 8) = 3.46). P21 male littermates were used in each individual experiment. The

levels of NPY-ir were measured in eight to ten sections from each animal. The open black circles

indicate the value from each individual animal. The black lines link the matched control and

stressed animals from each individual experiment.

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Figure 5-16. Injection per se did not alter the level of adrenal NPY-ir.

The level of NPY-ir was not changed in the adrenal medulla from the saline-injected

animals compared to uninjected controls (n = 2). P21 male littermates were used in each

individual experiment. The levels of NPY-ir were measured from eight to ten sections from each

animal. The open black circles indicate the value from each individual animal. The black lines

link the matched control and stressed animals from each individual experiment.

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Figure 5-17. Blocking the NPY Y1 or Y5 receptor did not affect the level of adrenal NPY-ir.

(A). Adrenal sections from a control animal and 24 hrs after the cold FST; Mice were

injected (i.p.) either with saline or BIBP3226. Scale bars 100 μm. (B). No difference in the level

of NPY-ir was detected in the adrenal medulla from the BIIE-injected animals compared to

saline-injected animals. P21 male littermates were used in two of the three independent

experiments, and one paired group were P21 female littermates. (C). No difference in the level of

NPY-ir was detected in the adrenal medulla from L125,804 (Y5 receptor antagonist)-injected

animals compared to saline-injected animals. P21 male littermates were used in two of the three

independent experiments, and one paired group were P21 female littermates. Data are mean ±

SEM, n = 3; *p < 0.05; one-way ANOVA (post-hoc Tukey’s paired comparison; for (B), F (3, 8)

= 1.95; for (C), F (3, 8) = 0.823). The levels of NPY-ir were measured in eight to ten sections

from each animal. The open black circles indicate the value from each individual animal. The

black lines link the matched control and stressed animals from each individual experiment.

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5.3 Discussion

5.3.1 NPY inhibits the expression of adrenal TH and NPY.

NPY and the catecholamines are the most abundant hormones expressed in the adrenal

medulla and they coexist in all mouse chromaffin cells. Although it is known that stress increases

both the levels of adrenal NPY (Hiremagalur and Sabban, 1995) and TH, a marker of

catecholamine biosynthesis (Sabban et al., 1995), the role of the stress-induced increase in

adrenal NPY was not clear.

NPY was reported to inhibit TH promoter activity in the SK-N-MC cell line through a Y1

receptor and to decrease TH synthesis in PC12 cells (Cavadas et al., 2006; McCullough and

Westfall, 1996). Using an in vivo approach I have shown that NPY inhibits the expression of

adrenal TH via a Y1 receptor.

Intraperitoneal injection of BIBP 3226 (a Y1 antagonist) and BIIE0246 (a Y2 antagonist)

were associated with increased levels of adrenal TH and NPY-ir respectively. Both BIBP 3226

and BIIE 0246 cannot cross the brain barrier (Doods et al., 1999; Doods et al., 1996; Shoblock et

al., 2010) so the intraperitoneal injection of these two antagonists underlies the effect of

peripheral NPY on inhibiting adrenal activity.

Adrenal catecholamine secretion is partially regulated through an inhibitory autocrine

feedback effect that is mediated by α2 adrenergic receptors expressed in chromaffin cells

(Lymperopoulos et al., 2007). In my experiments I found a complete overlap of NPY and TH in

all chromaffin cells and an expression of Y1, Y2 and Y5 receptors in the adrenal medulla.

Although I cannot exclude the involvement of NPY arising from peripheral sources other than the

adrenal medulla , it is likely that it is adrenal NPY that is responsible for regulating the expression

of adrenal TH and neuropeptide Y in an autocrine manner.

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5.3.2 NPY does not completely suppress the stress response.

The level of adrenal NPY was increased 24 hours after stress (chapter 4) and the level of

adrenal TH was also up-regulated at the same time point after stress (this chapter). Although NPY

was shown to suppress the expression of adrenal TH, the stress-induced increase in the level of

adrenal NPY did not block the elevation in the level of adrenal TH twenty four hours after stress.

This suggests that the effect of NPY in repressing the level of adrenal TH is a chronic effect.

Besides the inhibitory role of NPY on adrenal catecholamine synthesis, most studies have

reported that NPY can stimulate the secretion of catecholamines from chromaffin cells (Cavadas

et al., 2006; Cavadas et al., 2002; Cavadas et al., 2001). A coupled increase of secretion and

biosynthesis of the catecholamines in response to stress is required for the fight-or-flight response

and to compensate for the changes in physiology (Hamelink et al., 2002; Discussion 4.3.5). This

first step of the stress response may involve the stimulatory action of NPY on adrenal

catecholamine release. Then in the second step of the stress response, NPY appears to participate

in a negative feedback loop suppressing the synthesis of adrenal catecholamines and expression

of itself. This may be in order to prevent an overactivation of the adrenal medulla.

In contrast to its role in regulating the neuronal branch of the stress response, NPY is not

essential for adrenocortical function during the stress response. The effects of NPY on

glucocorticoid secretion in other studies are controversial. Several in vivo studies suggested that

NPY had no acute effect on the level of plasma glucocorticoids (Mazzocchi et al., 1996;

Mazzocchi and Nussdorfer, 1987). An in vitro study indicated an inhibitory role of NPY on

corticosterone secretion from adrenocortical cells (Malendowicz et al., 1990) whereas another in

vitro experiment revealed an stimulatory role of NPY on corticosterone release in adrenocortical

cells (Neri et al., 1990). My results indicate that NPY may be not involved in regulating

corticosterone secretion which fits with other in vivo studies (Mazzocchi et al., 1996; Mazzocchi

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and Nussdorfer, 1987) and is also consistent with the result that loss of NPY did not affect the

adrenocortical response during metabolic stress fasting (Erickson et al., 1997; Erickson et al.,

1996).

However I cannot exclude the possibility that stress increases plasma glucocorticoids in

NPY knockout mice to a different extent compared to that in wild type animals and it is not clear

whether loss of NPY affects the other two components of the HPA axis, the hypothalamus and

pituitary gland.

In addition mice were not handled before an exposure to the cold FST and control

animals were not handled at all since they were individually housed until sacrificed. Therefore the

memory of a stressful experience might contribute to the sustained increase in circulating

corticosterone that was observed in the stressed animals.

5.3.3 Possible mechanisms underlying the regulatory role of NPY in controlling the levels of

adrenal TH and NPY.

An in vitro SK-N-MC cell line study indicated that NPY downregulated TH promoter

activity via a Y1 receptor (Cavadas et al., 2006). All Y receptors are G-protein coupled receptors

and their activation usually causes inhibitory responses including the inhibition of cAMP

accumulation (Lin et al., 2004). Therefore blocking Y1 receptors is predicted to lead to an

increase in cAMP accumulation. The TH promoter region contains a perfect motif for a cAMP

response element (CRE) at -45 to -38 bp, so the transcription of the TH gene is potentially

stimulated by pathways activated by cAMP (Cambi et al., 1989; Kilbourne et al., 1992; Kim et al.,

1994; Sabban et al., 2006).

NPY also inhibits the expression of itself in the adrenal medulla via a different receptor,

the Y2 receptor. The NPY promoter binds CREB and the Y2 receptor is also a G-protein coupled

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receptor so the activation of a Y2 receptor is expected to inhibit cAMP accumulation (Titolo et al.,

2008). Therefore the mechanism underlying the NPY gene regulation may also involve a cAMP-

potentiated pathway.

However it is difficult to explain how distinct Y receptors on chromaffin cells regulate

NPY and TH by the same signaling pathway. For example why does the Y1 receptor not regulate

NPY and why does the Y2 receptor not modulate TH?

NPY regulated the secretion of catecholamines via a Y1 receptor-coupled mitogen-

activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathway (Rosmaninho-

Salgado et al., 2007a; Rosmaninho-Salgado et al., 2007b). Therefore it is possible that a different

signaling pathway rather than the classical cAMP-CREB pathway might be involved in regulating

the synthesis of TH via the Y1 receptor.

In the CNS it was reported that NPY mediated a presynaptic inhibition through the Y2

receptor and a postsynaptic inhibition through the Y1 receptor in the hypothalamus (Fu et al.,

2004). In the PNS the splanchnic nerve innervating the adrenal medulla is the presynaptic input. I

speculate that NPY may mediate presynaptic inhibition via the Y2 receptor by suppressing

acetylcholine release from the splanchnic nerve and thus inhibiting the expression of adrenal

NPY. This speculation is supported by evidence showing that NPY reduced the calcium current

and inhibited acetylcholine release from rat vagal afferent neurons (Wiley et al., 1990). NPY may

suppress the activity of chromaffin cells by postsynaptic inhibition through the adrenal Y1

receptor and thus inhibit TH expression in chromaffin cells. These suggestions are consistent with

the finding that the splanchnic nerve innervation is required for the stress-induced increase of

adrenal NPY transcription but not for the induction of adrenal TH during s restraint-induced

stress response (Hiremagalur et al., 1994; Hiremagalur et al., 1995).

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5.3.4 Working hypothesis of how adrenal NPY modulates adrenal activity in the stress

response.

Although I have shown that loss of NPY and blocking NPY receptors was associated

with an increase in the level of adrenal TH, the functional consequence of the stress-induced

increase in the level of adrenal NPY is still not clear. My proposed working model is (1). stress

increases both adrenal NPY and TH expression one day after stress as part of an acute stress

response; (2). an increased release of NPY due to increased adrenal NPY then represses the

expression of both adrenal TH and NPY as part of a chronic stress response (Figure 5-18 A). In

chapter 3.2.7, it was shown that an increased synthesis of NPY prohormone could lead to a

consequential increase in the secretion of NPY. Moreover recent data in our lab suggests that the

stress-induced upregulation of adrenal catecholamine synthesis leads to a consequential elevation

in catecholamine release. These results indicate that an increased synthesis of hormones could

result in an elevated level of secretion and thus support the second point in my working

hypothesis.

Adrenal NPY may participate in a negative feedback loop to maintain adrenal

homeostasis and to prevent a pathological activation of the fight-or-flight response during stress.

The physiological function of NPY in modulating the neuronal branch of the stress response may

parallel the role of glucocorticoids in negatively regulating the HPA axis (Figure 5-18 B).

To investigate the functional consequences of the stress-induced increase in the level of

adrenal NPY, two methods including denervation of the adrenal gland by splanchnicectomy and

injection of a nicotinic receptor antagonist chlorisondamine could be used. These would

potentially block the stress-induced increase in the level of adrenal NPY (Hiremagalur et al., 1994;

Hiremagalur et al., 1995). Possible future research would be to compare the level of adrenal TH

and other sympathetic parameters such as heart rate, blood pressure and thermogenesis in stressed

intact and stressed denervated animals.

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5.4 Conclusion

I conclude that NPY negatively regulates the expression of adrenal TH and NPY. NPY

plays an important role in inhibiting the neuronal branch of the stress response but does not affect

the adrenocortical response during stress.

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Figure 5-18. Working model.

(A). A working model depicting the role of adrenal NPY in modulating adrenal

functioning during the stress response. (B). NPY may function in a negative feedback loop

regulating the activation of the sympatho-adrenal system during the stress response.

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Chapter 6

Discussion

In this thesis I first described experiments that determined how a functional single

nucleotide polymorphism (T1128) in the NPY signal sequence altered NPY prohormone

biosynthesis and secretion (chapter 3). This SNP is associated with metabolic and cardiovascular

disease and humans containing this SNP have higher levels of plasma NPY after exercise

(Pesonen, 2006, 2008).

Plasma NPY is thought to originate from post-ganglionic sympathetic neurons, since

simulation of these neurons will lead to a secretion of NPY into the blood (Kuo et al., 2007a;

Morris et al., 1986). In the remainder of my thesis research I have studied the regulation of NPY

synthesis in the sympathetic nervous system (SNS) using chromaffin cells (modified post-

ganglionic neurons) and the adrenal medulla as a model system. I have shown that stress

increases the activity of chromaffin cells and that the expression of adrenal NPY is up-regulated

by two different stressors (chapter 4). NPY receptors are found in the heart, smooth muscle cells,

fat tissue, pancreas and the adrenal gland (Dumont et al., 1992), so peripheral NPY is thought to

regulate both the cardiovascular system and metabolism (Kuo et al., 2007a; Kuo et al., 2008).

In the peripheral nervous system NPY post-synaptically stimulates, and presynaptically

inhibits, norepinephrine release from sympathetic nerves (Kuo et al., 2007a) , and my data

(chapter 5) as well as the results from other studies suggests that NPY regulates both the secretion

and synthesis of the catecholamines in chromaffin cells (Cavadas et al., 2006; Cavadas et al.,

2002; Cavadas et al., 2001) . The catecholamines are importantly involved in modulating the

cardiovascular system (Lymperopoulos et al., 2007) and metabolism (Bachman et al., 2002;

Bowers et al., 2004).

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Therefore in this chapter I will discuss the role of NPY in both the cardiovascular system

and in metabolic regulation. Besides the function of peripheral NPY in these two systems, I will

outline how the role of NPY in the central nervous system is coordinated with the SNS to regulate

the cardiovascular system and metabolism. I will also discuss the role of NPY in the stress

response. In particular I will discuss the function of NPY in controlling the cardiovascular system

during stress and the role of peripheral NPY in stress-induced metabolic syndrome.

6.1 Role of NPY in the cardiovascular system

6.1.1 Role of peripheral NPY in cardiovascular modulation

The first identified biological activity of NPY was to induce a prolonged vasoconstriction

and this effect was resistant to α-adrenergic receptor blockage (Lundberg and Tatemoto, 1982).

Later it was determined that theY1 receptor mediated this process since in Y1 knockout mice, the

NPY-induced increase in blood pressure was abolished (Pedrazzini et al., 1998).

Indirectly NPY enhanced norepinephrine-evoked vasoconstriction in an intracellular

calcium-dependent manner, and the effect of NPY application was through a post-synaptic Y1

receptor (Abe et al., 2010; Fallgren et al., 1993; Kuo et al., 2007a; Pedrazzini et al., 1998). NPY

also suppressed norepinephrine release by a presynaptic Y2 receptor via a negative feedback

mechanism (Donoso et al., 1988; Kuo et al., 2007a; Maynard and Burnstock, 1994). An

interaction between the NPY system and catecholamine signaling is thus important in controlling

the cardiovascular system.

The role of peripheral NPY in elevating blood pressure is consistent with the finding that

the T1128C SNP in the NPY preprohormone is associated with a higher risk of heart disease

(Pesonen, 2006; 2008).

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6.1.2 Role of the adrenal medulla in cardiac function

Chromaffin cells in the adrenal medulla are modified post-ganglionic sympathetic

neurons and are part of the sympathetic nervous system. Chromaffin cells can fire action

potentials and are innervated by preganglionic sympathetic fibers. Nicotinic acetylcholine

receptors expressed by chromaffin cells are activated by acetylcholine released from

preganglionic nerves leading to depolarization of the chromaffin cells. Chromaffin cells

synthesize both NPY and the catecholamines (Whim, 2006). They are the major source of plasma

epinephrine. Upon depolarization the catecholamines and NPY are released into the blood

(Axelrod and Reisine, 1984; Whim, 2006).

As a source of plasma NPY and the catecholamines, the adrenal medulla is expected to

have a role in cardiac function. A transcriptome study in a genetic hypertension and hypotension

mouse model showed that mRNA levels of adrenal NPY and TH (a marker of catecholamine

synthesis) were significantly higher in the hypertensive mouse strain compared with the

hypotensive strain (Fries et al., 2004).

Direct evidence that the adrenal medulla modulates cardiac functions comes from a study

of adrenal G protein-coupled receptor kinase 2 (GRK2) upregulation in mediating a sympathetic

overdrive that promotes heart failure (Lymperopoulos et al., 2007). Hyperactivity of the

sympathetic nervous system and increased catecholamine release was found in two heart failure

models. These overactive responses were caused by an upregulation of adrenal GRK2. The

increase of adrenal GRK2 disrupted α2-adrenergic receptor (α2AR) signaling in the adrenal

medulla and this disruption impaired the autocrine feedback inhibition of catecholamine secretion

by α2AR’s in chromaffin cells (Lymperopoulos et al., 2007).

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6.1.3 Role of central NPY in the cardiovascular system

Central injection of NPY induced hypotension in rat (Fuxe et al., 1983). This is the

opposite effect to the vasoconstriction that is produced by peripheral NPY injection in rats

(Lundberg and Tatemoto, 1982). A transgenic rat line in which NPY is overexpressed under the

control of its natural regulatory elements has been developed. These animals showed decreased

blood pressure and reduced catecholamine release suggesting that the role of endogenous NPY is

a long-term inhibition of the sympathetic nervous system (Michalkiewicz et al., 2003). The

central NPY vasodepressor effect was primarily mediated by a presynaptic Y2 receptor inhibition

and thus a decreased sympathetic outflow to the heart (Chen and Westfall, 1993). This conclusion

is further supported by evidence that the heart rate was increased in NPY Y2-deficient mice

(Naveilhan et al., 1999).

6.2 Role of NPY in metabolic regulation

6.2.1 Role of central NPY in feeding

NPY has been noted for its stimulatory effect on feeding (Lin et al., 2004). Central

administration of NPY significantly increases food intake and chronic infusion of NPY can

induce obesity from overeating (Flier and Maratos-Flier, 1998).

The first conventional NPY knockout mouse on a 129/SvCp-J background did not show

a phenotype of reduced feeding, body weight or adiposity under normal conditions (Erickson et

al., 1996). This report was surprising since for decades neuroscientists believed the central role of

NPY was in promoting hyperphagia but this NPY knockout mouse line had no phenotype with

respect to body weight regulation (Dube et al., 1994; Levine and Morley, 1984; Stanley et al.,

1986). However when the NPY knockout mice were backcrossed onto a C57BL/6 background,

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they showed decreased re-feeding after fasting. This result suggested an altered behavior of food

intake in the mice lacking NPY (Bannon et al., 2000). The lack of a clear phenotype related to

energy balance in NPY germline knockout mice might be explained by compensatory

mechanisms in development (Trivedi et al., 2001).

Y1 knockout mice display slightly diminished feeding and strongly reduced fasting-

induced re-feeding, however body fat and serum leptin was increased in these mice (Pedrazzini et

al., 1998). Y5-deficent mice display normal food intake and body weight but developed obesity

when they were older than 30 weeks (Marsh et al., 1998). The data generated from these

knockout mice suggested that the Y1 and Y5 receptors were important for maintaining energy

homeostasis.

One Y2 knockout mouse model displayed decreased bodyweight gain (Sainsbury et al.,

2002b) whereas another Y2 knockout mouse line showed increased body weight as well as food

intake (Naveilhan et al., 1999). The deletion of Y2 receptors in the adult mouse hypothalamus led

to transiently decreased body weight and increased food intake indicating the functional role of

the hypothalamic Y2 receptors in controlling feeding. This also illustrates the importance of site-

specific knockout models compared to a conventional knockout, as developmental and

compensation mechanisms may mask such effects in germline knockout mice (Sainsbury et al.,

2002a).

6.2.2 Role of peripheral NPY in metabolic regulation

Compared to the studies of hypothalamic NPY, less attention has been given to the role

of extrahypothalamic NPY in metabolic regulation. Peripheral NPY is suggested to be a marker

of sympathetic nervous system activity (Dumont et al., 1992; Heilig, 2004). NPY and the

catecholamines are co-released from sympathetic neurons and both transmitters regulate the

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activity of peripheral tissues including the heart, liver, pancreas, adrenal glands and adipose tissue

since these are innervated by the sympathetic nervous system (Heilig, 2004; Kuo et al., 2007a).

Transgenic mice overexpressing NPY in adrenergic and noradrenergic neurons (i.e. brain

noradrenergic neurons and peripheral sympathetic neurons) showed an increase of adiposity,

impaired glucose tolerance and liver triglyceride accumulation (Ruohonen et al., 2008).

Humans carrying the T1128C polymorphism in the NPY preprohormone have higher

levels of plasma NPY after exercise (Kallio et al., 2001). Our studies suggest that this SNP

augments the packaging of the NPY prohormone into DCSGs and thus led to an up-regulation of

NPY secretion (Mitchell et al., 2008). This data may suggest a molecular mechanism underlying

the observation that a higher level of exercise-induced plasma NPY was found in the T1128

carriers (Kallio et al., 2001) and that the SNP was associated with higher levels of serum

cholesterol and increased risk for metabolic disease (Karvonen et al., 1998; Karvonen et al., 2001;

Pesonen, 2006, 2008). These phenotypes observed in humans with this SNP are also consistent

with the role of NPY in mediating stress-induced obesity (Kuo et al., 2007b). However the role

of NPY in regulating lipid metabolism is complex since it also inhibits lipolysis (Heilig et al.,

2004; Nordman et al., 2005).

6.3 Role of NPY in the stress response

6.3.1 Role of NPY in controlling the two major stress pathways in the PNS

One characteristic of the stress response is the activation of the HPA axis leading to a

marked secretion of the glucocorticoids into the blood (Axelrod and Reisine, 1984; Sapolsky et

al., 2000). In my experiments the cold FST induced-stress led to an increased level of plasma

corticosterone (CORT) even 24 hours after the stress and this effect was maintained in the NPY

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knockout mice (chapter 5). An increase in plasma CORT was also observed in both the NPY

deficient mice and wild type animals during the response to acute fasting (Erickson et al., 1997;

Erickson et al., 1996). Furthermore plasma CORT was elevated during restraint or cold stress in

the conditional NPY transgenic animals (Ruohonen et al., 2009).These findings suggest that NPY

is not essential for an appropriate functioning of the adrenal cortex during stress.

Besides the HPA axis, sympathetic activity is enhanced in the stress response and NPY is

released from the sympathetic nerves (Kuo et al., 2007a). In my experiments, I found that NPY

expression in the adrenal medulla was elevated by diverse stressors including exposure to fox

urine, cold FST and a metabolic stress, food restriction (data not shown). In addition peripheral

NPY is a negative regulator of adrenal NPY expression and catecholamine synthesis indicating

that NPY is involved in inhibiting neuronal activation during the stress response.

6.3.2 Peripheral NPY mediates stress-induced hypertension

Stress activates the sympathetic nervous system leading to a secretion of NPY from

sympathetic nerves, and produces hypertension which can be blocked by a Y1 antagonist (Han et

al., 1998). This study indicated that NPY is a mediator of stress-induced hypertension.

This hypothesis was further supported by a study using conditional NPY transgenic mice

(Ruohonen et al., 2009). Transgenic mice with an overexpression of NPY in catecholaminergic

neurons showed an increase in arterial pressure during the night after surgery stress but no

difference during the recovery phase (Ruohonen et al., 2009). This result suggests that

overexpression of NPY in catecholaminergic neurons increases stress-induced sympathetic

activity and hypertension.

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6.3.3 Antagonistic effect of central and peripheral NPY in the cardiac response may modify

the stress response

The data from two NPY transgenic models, both of which over-express NPY was found

to be contradictory (Michalkiewicz et al., 2003; Ruohonen et al., 2009). The main difference

between these two transgenic models was (1). excess NPY was expressed in numerous brain

regions including the hypothalamus which regulates the sympathetic output to the peripheral

system in the conventional transgenic rats. (2). however NPY was only over-expressed in brain

noradrenergic neurons and in the sympathetic nervous system in the conditional transgenic mice.

It is possible that the overexpression of NPY in the transgenic rats inhibited the sympathetic drive

by a CNS-mediated mechanism whereas the increased sympathetic activity in the NPY transgenic

mice was mainly due to the actions of peripheral NPY.

The results in the two different NPY transgenic models are reminiscent of the opposite

effects from the peripheral and central injections of NPY which increased and decreased blood

pressure, respectively. The data suggests a central inhibitory, but peripheral stimulatory, action of

NPY in regulating the sympathetic outflow (Kuo et al., 2007a).

The antagonistic effects of central and peripheral NPY in controlling sympathetic outflow

to the heart might be a survival mechanism in response to stress such that the increased

vasoconstriction by peripheral NPY during stress is an adaptive response, whereas repression of

the sympathetic drive by central NPY would prevent a pathological activation.

6.3.4 Interaction between NPY and catecholamines in cardiovascular modulation may be a

survival mechanism in response to stress

Many in vitro studies have investigated the effects of NPY on catecholamine release and

synthesis in chromaffin cells. The catecholamines and NPY are co-released (Whim, 2006). Most

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studies reported that NPY stimulated catecholamine secretion from chromaffin cell cultures

(Cavadas et al., 2002; Cavadas et al., 2001). This effect involved the Y1 receptor since the NPY-

induced release of the catecholamines was abolished in chromaffin cells from Y1-deficient mice

(Cavadas et al., 2006).

The basal level of catecholamine synthesis in the adrenal medulla was up-regulated in Y1

knockout mice (Cavadas et al., 2006). The inhibitory role of NPY in adrenal catecholamine

synthesis is further supported by my results showing that the expression of TH in the adrenal

medulla was also basally activated in the NPY knockout mice (chapter 5). Moreover I found that

i.p. injection of BIBP3226 (a Y1 antagonist) and BIIE0246 (a Y2 antagonist) led to an increase in

the levels of adrenal TH and NPY respectively (chapter 5). Since neither of the antagonists cross

the brain barrier (Doods et al., 1999; Doods et al., 1996; Shoblock et al., 2010), my data suggests

that the role of NPY in the periphery is to directly down-regulate the levels of adrenal

catecholamines and NPY.

My speculation for explaining the stimulatory action of NPY on catecholamine release

and the inhibitory effect of NPY on the synthesis of adrenal catecholamines is as following: (1)

an initial up-regulation of catecholamine signaling by NPY and the cooperative effects of these

transmitters in enhancing the cardiac system are adaptive processes triggered by the fight-or-

flight response during stress; (2). as the stress-induced activation of the sympathetic and

cardiovascular systems proceeds, NPY participates in a negative feedback loop that represses the

synthesis of the catecholamines and also of NPY. The function of this loop may be to prevent the

system from becoming maladaptive due to an over-activation of the stress response.

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6.3.5 Peripheral NPY mediates stress-induced obesity

Stress exaggerated the high fat diet-induced obesity which was mediated by peripheral

NPY signaling (Kuo et al., 2007b). Stress increased the release of NPY and glucocorticoids from

sympathetic nerves, and the adrenal cortex, respectively. In a glucocorticoid-dependent manner,

the expression of sympathetic NPY and Y2 receptors in the white adipose tissue (WAT) were up-

regulated by stress. The activation of the Y2 receptor stimulated the growth of abdominal fat

leading to metabolic syndrome (Kuo et al., 2007b). The data above not only suggest a direct

effect of the peripheral NPY in modulating metabolism but also link stress with obesity in

peripheral NPY/ Y2 signaling.

Stress activates the sympatho-adrenomedullary system stimulating the secretion of

epinephrine and to a lesser extent, norepinephrine, into the blood. This process is fundamental for

the fight-or-flight response. By activating β adrenergic receptors, the catecholamines enhance

lipolysis as well as inhibiting adipocyte proliferation in the WAT, and stimulate thermogenesis in

the brown fat tissue (Bachman et al., 2002; Bowers et al., 2004). In my experiments injection (i.p.)

of BIBP3226 (a Y1 antagonist) was associated with an increase in the levels of adrenal TH (a

marker of catecholamine synthesis). BIBP3226 does not cross the brain-barrier (Doods et al.,

1996; Shoblock et al., 2010), so the inhibitory effect of NPY on catecholamine synthesis in the

adrenal medulla was mediated by a peripheral mechanism. Since catecholamine signaling

enhances energy expenditure, peripheral NPY may do the opposite by slowing down metabolism

not only via Y2 signaling directly but also by negatively regulating catecholamine synthesis.

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6.4 Future directions

In my current study I have demonstrated that the cold FST leads to a long-term increase

of adrenal NPY synthesis. A metabolic stress, food restriction, also elevates the levels of adrenal

NPY (data not shown). In future work I hope to directly investigate the role of adrenal NPY in

altering metabolism and the cardiovascular response to stress

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Appendix A

Appendix A. The cold FST did not alter the level of adrenal NPY-ir in vitro or in situ 30 min

after stress.

(A). The level of NPY-ir was not altered in chromaffin cells 30 min after stress compared with

the control (n = 1). The levels of NPY-ir were measured in seventy cells from each animal (N.S.,

not significant). (B). The level of NPY-ir was not changed in adrenal sections 30 min after stress

compared with the control (n = 1). The levels of NPY-ir were measured in eight sections from

each animal (N.S., not significant).

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Appendix B

Appendix B. The level of adrenal NPY-ir was increased in epinephrine-injected animals and

epinephrine-treated acute slices.

(A). The level of NPY-ir in adrenal frozen sections was increased 24 hours after epinephrine

injection compared with saline injection (n=2). The levels of NPY-ir were measured in eight

sections from each animal (p < 0.05 in each individual experiment). (B). NPY-ir was increased 24

hours after treating 400 μm thick acute adrenal slices with 100 mM epinephrine (n=1). Methods:

20 μm frozen adrenal sections were prepared by re-sectioning the treated acute slices. The levels

of NPY-ir were measured in eight sections from each animal (p < 0.05).

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Appendix C

(A). Phosphate buffered salt solution (PBS), pH 7.4

NaCl: 137 mM

KCl: 2.7 mM

Na2HPO4.7H2O: 10 mM

KH2PO4: 1.8 mM

(B). Blocking solution (0.25%)

PBS: 20 mL

Bovine serum albumin (BSA): 0.05 g

Triton X-100: 10 μl

(C). HBSS+20mM HEPES (20H HBSS), pH 7.25

KCl: 5.3 mM

KH2PO4: 0.44 mM

NaHCO3: 4 mM

NaCl: 138 mM

Na2HPO4.7H2O: 0.3 mM

Glucose: 5.5 mM

HEPES: 20 mM

(D). Trituration medium

HBSS: 10 mL

159

BSA: 1 mg/ml BSA

MgCl2: 8 mM

(E). Collagenase solution

Collagenase: 1 mg

BSA: 6 mg

20H HBSS: 1 mL

(F). Trypsin solution

Trypsin: 1 mg

BSA: 6 mg

20H HBSS: 1 mL

(G). 10 X Tris-Glycine (1 X Tris-Glycine; SDS-PAGE running buffer)

Tris base: 15.14 g

Glycine: 72.07 g

SDS: 5 g

To final volume of 500 mL

(H). 5 X Transfer buffer

Tris base: 7.5 g

Glysine: 35.625 g

To final volume of 500 mL

(I) 1 X transfer buffer

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5 X transfer buffer: 100 mL

ddH2O: 300 mL

Methanol: 100 mL

(J). 10 X PBS

NaCl: 40 g

KCl: 1 g

KH2PO4: 1 g

Na2HPO4.7H2O: 10.8 g

To a final volume of 500 mL

(K) 1 X PBS-T (Western blot washing buffer)

10 X PBS: 50 mL

ddH2O: 450 mL

Tween-20: 0.3%

161

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Vita

Qian Wang

Education Ph.D. Candidate in Cell and Developmental Biology Sep 2006-Present

The Pennsylvania State University, USA

Dissertation Regulation of neuropeptide Y expression and the sympathoadrenal response to

stress

Bachelor of Science, major in Biotechnology Sep 2002- Jul 2006

Shanghai Jiao Tong University, P.R.China

Publications, Abstracts and Manuscripts

Wang Q and Whim MD. Acute stress leads to a long-term change of adrenal neuropeptide

Y. Manuscript in preparation, 2011.

Wang Q and Whim MD. Neuropeptide Y inhibits the neuronal branch of the stress

response. Manuscript in preparation, 2011.

Ramamoorthy P, Wang Q, and Whim MD. Cell type-dependent trafficking of neuropeptide

Y- containing dense core granules in CNS neurons. Under revision, 2011.

Wang Q and Whim MD. Acute stress leads to a long-term change in the adrenal levels of

neuropeptide Y (2010). Annual meeting of Society for Neuroscience.

Wang Q and Whim MD. Acute stress alters the levels of neuropeptide Y in a

sub-population of adrenal chromaffin cells (2009). Annual meeting of Society for

Neuroscience.

Wang Q, Mitchell GC, Ramamoorthy P, Whim MD (2008). A single nucleotide

polymorphism associated with a change in serum cholesterol levels increases synthesis and

secretion of neuropeptide Y. Annual meeting of Society for Neuroscience.

Mitchell GC*, Wang Q*, Ramamoorthy P, Whim MD (2008). A common single

nucleotide polymorphism alters the synthesis and secretion of neuropeptide Y. J Neurosci.

28:14428-14434. * Co-first author.

Honors and Awards

2009 J Ben and Helen D. Hill Memorial Fund Award

2006 Huck Institutes of Life Sciences Fellowship, PSU